WO2021184560A1 - 一种以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗 - Google Patents

一种以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗 Download PDF

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WO2021184560A1
WO2021184560A1 PCT/CN2020/096024 CN2020096024W WO2021184560A1 WO 2021184560 A1 WO2021184560 A1 WO 2021184560A1 CN 2020096024 W CN2020096024 W CN 2020096024W WO 2021184560 A1 WO2021184560 A1 WO 2021184560A1
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ncov
vector
cells
recombinant adenovirus
adenovirus
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French (fr)
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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
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中国人民解放军军事科学院军事医学研究院
康希诺生物股份公司
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Priority to US17/606,792 priority Critical patent/US20230022109A1/en
Priority to EP20925416.8A priority patent/EP3950947A4/en
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Definitions

  • the present invention relates to a recombinant novel coronavirus vaccine, which aims to prevent the epidemic situation of the novel coronavirus.
  • the invention belongs to the technical field of bioengineering.
  • 2019-nCoV-2 The 2019 novel coronavirus, SARS-CoV-2 (also known as 2019-nCoV), was named by the World Health Organization on January 12, 2020. It is a new strain of coronavirus that has never been found in humans before. This virus is the seventh coronavirus (CoV) that can infect humans.
  • the incubation period of human infection with 2019-nCoV is generally 1 to 14 days. Common signs after infection with 2019-nCoV include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death.
  • March 17, 2020 my country has reported 81,116 confirmed cases and 3,231 deaths; other countries in the world have reported 98,486 cases, with a total of 3848 deaths, with an average mortality rate of 3.94%.
  • the new type of coronavirus belongs to the ⁇ genus of coronaviruses. It has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm.
  • Phylogenetic analysis of the whole genome of the virus shows that the virus is most closely related to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) previously found in Chinese bats (89.1% nucleotide similarity) ).
  • 2019-nCoV has the closest relationship with bat coronavirus bat-SL-CoVZC45, with an amino acid identity of 82.3%, and an amino acid identity with SARS-CoV of about 77.2%.
  • the receptor that 2019-nCoV binds when entering human cells is the same as that of SARS-CoV, both of which are angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • Coronavirus is a large family of viruses. So far, the main causes of human infection outbreaks are SARS-CoV, MERS-CoV and SARS-CoV-2. There are not many reports on the structure, function, key infection targets, mechanism of action, and vaccine development of SARS-CoV-2. Many studies mainly focus on SARS-CoV and MERS-CoV vaccines. The main types of research and development include viral vector vaccines, Nucleic acid vaccines, subunit vaccines, virus-like particle vaccines, inactivated vaccines and live attenuated vaccines. The target antigens of recombinant coronavirus vaccines are generally in the form of full-length S protein and truncated S protein (S1 and RBD).
  • the purpose of the present invention is to provide a recombinant new coronavirus vaccine.
  • the present invention first provides a polynucleotide for encoding the S protein gene optimization of a novel coronavirus vaccine, and the sequence of the polynucleotide is shown in SEQ ID NO:1.
  • the polynucleotide uses the replication-deficient human type 5 adenovirus with combined deletion of E1 and E3 as a vector, and uses HEK293 cells integrating the E1 gene of the adenovirus as a packaging cell line to obtain a recombinant adenovirus vector new coronavirus vaccine through packaging.
  • the present invention also provides a vector containing the above-mentioned polynucleotide.
  • the vector is pDC316.
  • the present invention also provides a human replication-deficient recombinant adenovirus capable of expressing the polynucleotide.
  • the recombinant adenovirus is derived from the AdMax adenovirus system.
  • the invention also provides the application of the above-mentioned recombinant adenovirus in the preparation of a vaccine for the prevention of novel coronavirus pneumonia.
  • the recombinant adenovirus is prepared as an injection, nose drops or spray.
  • the recombinant adenovirus is prepared as an intramuscular injection.
  • the present invention provides a method for preparing the above-mentioned recombinant adenovirus capable of expressing the S protein of the novel coronavirus, and the method includes the following steps:
  • the shuttle plasmid vector in step (1) is pDC316.
  • the backbone plasmid in step (2) is pBHGlox_E1, 3Cre, both of which belong to the AdMax adenovirus system, and are used together for packaging recombinant adenovirus containing polynucleotides encoding the new coronavirus S protein in host cells .
  • the cells in step (3) are HEK293 cells.
  • the expanded culture method in step (5) is suspension culture.
  • the purification method in step (6) is Source 30 Q chromatography.
  • the recombinant adenovirus capable of expressing the new coronavirus S protein provided by the present invention is used as a new coronavirus vaccine (Ad5-nCoV), which has good immunogenicity in both mouse and guinea pig models, and can induce the body to produce in a short time Strong cellular and humoral immune response.
  • Ad5-nCoV coronavirus vaccine
  • the protective effect study on hACE2 transgenic mice showed that a single immunization with Ad5-nCoV can significantly reduce the viral load in the lung tissue after 14 days. It shows that the vaccine has a good immune protection effect against the 2019 new coronavirus.
  • the vaccine preparation method is quick and simple, and can realize large-scale production in a short period of time for responding to sudden epidemics.
  • FIG. 1 The Western blot identification map of the target antigen expression of the primary virus species of the recombinant novel coronavirus vaccine candidate strain
  • FIG. 7 Comparison of serum antibody levels induced by different immunization methods on the 14th day of mouse immunization with recombinant adenovirus
  • Figure 10 Representative diagram of cellular immune response in the intramuscular injection group
  • Figure 11 Representative diagram of cellular immune response in the nasal drip immune group
  • Figure 12 Representative graph of cellular immune response in control group
  • Figure 14 Comparison diagram of immunogenicity detection of Ad5-nCoV guinea pig model
  • FIG. 1 Comparison of viral load in lung tissue of mice after SARS-CoV-2 challenge.
  • Example 1 Preparation of recombinant new coronavirus vaccine using human replication-deficient adenovirus as a vector
  • the target antigen of the recombinant novel coronavirus vaccine is the S protein of the novel coronavirus strain (Genebank number: NC_045512.2).
  • S protein of the novel coronavirus strain Genebank number: NC_045512.2
  • Upgene software (Gao, W. Rzewski, A. Sun, H. Robbins, PD& Gambotto, A. UpGene: Application of a web-based DNA codon optimization algorithm. Biotechnol Prog, 2004.20(2): p.443- 8.) Perform codon optimization and change most of the rare codons of the S protein gene to high frequency codons. Secondly, considering that software optimization may mechanically change the codons to the most frequently used codons, the efficiency of protein translation may not be significantly improved due to the influence of tRNA usage efficiency and mRNA secondary structure.
  • the content of rare codons (used frequency ⁇ 70%) of the new coronavirus S protein gene was 34%, the content of high frequency codons (used frequency> 90%) was 23%, and the GC content was 36% .
  • the content of rare codons was reduced to 3%, the content of high-frequency codons was increased to 81%, and the GC content was increased to 58%.
  • the homology with the original S protein gene sequence was 70.4%.
  • the Kozak sequence was added before the translation initiation codon, and the restriction site EcoRI was inserted upstream of the entire sequence, and the restriction site HindIII was inserted downstream to synthesize the gene sequence.
  • the original sequence of the S protein gene was synthesized. See SEQ ID NO:1 for the optimized sequence of the S protein gene (enzyme cutting sites are EcoRI and HindIII), and see SEQ ID NO: 2 for the original gene sequence of the S protein (enzyme cutting sites are SmaI and SalI).
  • the above synthesized gene sequence was digested with HindIII and SalI, or SmaI and SalI, respectively, and the target gene fragment was recovered and ligated to the shuttle plasmid pDC316 of the AdMax adenovirus system (Microbix Biosystems Inc., Canada) to transform DH5- Alpha-competent, spread Amp r LB plates, pick a single clone for colony PCR identification, and sequence the clones that are identified as positive by PCR.
  • the plasmid whose S protein gene sequence has not been optimized is denoted as pDC316-nCoV_S
  • the plasmid optimized by the S protein gene is denoted as pDC316-nCoV_Sopt.
  • the plasmid map of pDC316-nCoV_Sopt is shown in Figure 1.
  • the 6 shuttle plasmids and pDC316 vector constructed above were transfected into HEK293 cells using the transfection reagent TurboFect Transfection Reagent (Thermo Scientific, REF, R0531), and the cells were harvested for WB detection after 48 hours.
  • the experimental method is as follows:
  • HEK293 cells were seeded in a 6-well plate at 8 ⁇ 10 5 cells/well and cultured overnight in a 37°C, 5% CO 2 cell incubator.
  • the medium was changed to fresh DMEM medium containing 2% FBS, 2 mL per well.
  • transfection take 2 ⁇ g of the corresponding plasmid from each transfection well, add it to 200 ⁇ L of FBS-free DMEM medium, mix well, add 3 ⁇ L of transfection reagent, mix gently, and leave it at room temperature for 15 minutes.
  • the cells were cultured in a 37°C, 5% CO 2 cell incubator. After 5 hours, the medium was changed to fresh DMEM medium containing 10% FBS. After 48 hours, the cells were collected, samples were prepared, and WB detection was performed.
  • Sample preparation 48 hours after transfection, carefully aspirate and discard the medium, resuspend the cells in PBS, centrifuge at 500g for 5 minutes, and discard the clear.
  • Cells were resuspended in 200 ⁇ L RIPA buffer (Thermo Scientific, Prod#89900, plus appropriate amount of protease inhibitors and nucleases), ice-bath for 15 minutes, centrifuged at 4°C and 12000rpm for 5 minutes, take the supernatant, add 1/3 volume containing 200mmol /L DTT 4 ⁇ SDS-PAGE loading buffer, heated at 95°C for 5 minutes, frozen, and used for WB detection.
  • RIPA buffer Thermo Scientific, Prod#89900, plus appropriate amount of protease inhibitors and nucleases
  • Western blot detection use 10 wells of 4-20% SDS-PAGE gradient gel for SDS-PAGE, with a loading volume of 30 ⁇ L per well. Electrophoresis conditions: 80V, 15min; 180V, until the bromophenol blue just came out of the gel.
  • the protein on the SDS-PAGE gel was transferred to the nitrocellulose membrane by a electroporation device, and the electroporation condition was 300mA for 1 hour. After the electroporation was completed, the nitrocellulose membrane was blocked with 5% skimmed milk powder for 1 hour, and then anti-S protein rabbit polyclonal antibody (Yiqiao Shenzhou, catalog number 40150-T52) was added at a dilution of 1:2000, and placed overnight at 4°C.
  • ⁇ -actin was used as the sample to test the internal control, and the results are shown in Figure 2.
  • 1 transfected pDC316-nCoV_S cells
  • 2 transfected pDC316-nCoV_tPA-S cells
  • 3 transfected pDC316-nCoV_oriSIP-Sopt cells
  • 4 Transfection of pDC316-nCoV_Sopt cells
  • 5 transfection of pDC316 empty vector cells
  • 6 pre-transfection of Marker
  • 7 transfection of pDC316 empty vector cells
  • 8 transfection of pDC316-nCoV_S1 cells
  • 9 transfection of pDC316-nCoV_S1opt cells.
  • the vectors pDC316-nCoV_Sopt, pDC316-nCoV_oriSIP-Sopt and pDC316-nCoV_S1opt constructed above were co-transfected with the backbone plasmid pBHGlox_E1,3Cre of the AdMax adenovirus system to HEK293 cells for packaging of recombinant adenovirus.
  • the process is as follows:
  • Ad5-nCoV_Sopt The virus species expressing the optimized sequence of the full-length protein gene of the tPA signal peptide S are labeled as Ad5-nCoV_Sopt, and the virus species expressing the optimized sequence of the full-length protein gene of the original signal peptide S is labeled as Ad5-nCoV_oriSIP-Sopt, which expresses the tPA signal peptide S1 protein gene.
  • Ad5-nCoV_oriSIP-Sopt The virus seed of the optimized sequence is labeled Ad5-nCoV_S1opt.
  • the primer sequence is as follows:
  • the PCR amplification conditions are:
  • HEK293 cells were infected with different recombinant adenoviruses. After 48 hours, the cells were collected for Western blot detection of the target antigen.
  • the three virus species Ad5-nCoV_Sopt, Ad5-nCoV_oriSIP-Sopt and Ad5-nCoV_S1opt can all detect the obvious expression of the target protein. The results are shown in Figure 3, where 1: Pre-stained Marker; 2 and 3: Ad5-nCoV_oriSIP-Sopt infected cells; 4 and 5: Ad5-nCoV_Sopt; 6: Ad5-nCoV_S1opt infected cells. 7 and 8: Blank cells.
  • HEK293 cells were cultured in suspension at 120 rpm at 37°C and 5% CO 2. During seed inoculation, dilute cells with an activity greater than 95% to 1.0 ⁇ 10 6 cells/mL, with a final volume of 300 mL. The P5 generation recombinant adenovirus was infected with HEK293 cells at MOI 10, and cultured at 37°C, 5% CO 2 , and shaking at 130 rpm. Samples were taken every 24 hours to detect cell activity and density.
  • the cell shake flask was placed in a -70°C refrigerator and 37°C water bath for repeated freezing and thawing twice.
  • Benzonase (20U/mL) was added and enzymatically hydrolyzed in a water bath at 34-36°C for 2 hours. Centrifuge at 12000g for 10 minutes, collect the virus-containing supernatant, and discard the precipitate.
  • the experimental method and process are the same as 3.2.1.
  • the results of agarose gel electrophoresis showed that all three virus species could be amplified to a single band of interest, and the fragment size was correct.
  • the target band was recovered by gel and sequenced, and the comparison result showed that the sequence of the sequencing result was completely correct.
  • Clontech Adeno-X TM Rapid Titer Kit was used for the determination of recombinant adenovirus titer. The operation is carried out according to the instructions attached to the kit, and the specific methods are as follows:
  • HEK293 cells Inoculate HEK293 cells in a 24-well plate.
  • the cell density is 5 ⁇ 10 5 cells/mL, 0.5 mL is inoculated per well, and the medium is MEM+10% FBS.
  • the cells were cultured in a 37°C, 5% CO2 incubator for 48 hours.
  • Infection titer number of positive cells in the field ⁇ number of fields per well / (virus volume (mL) ⁇ dilution)
  • the results of titer determination showed that after the purified recombinant adenovirus was concentrated, the infection titer reached 1.0 ⁇ 10 10 ifu/mL or more.
  • the results of the determination of the number of virus particles showed that the purified recombinant adenoviruses reached more than 1.0 ⁇ 10 11 VP/mL after concentration.
  • mice 100 SPF female BALB/c mice (6-8 weeks old) were randomly divided into 10 groups, each with 10 mice. According to the grouping situation shown in Table 1, the mice were immunized with Ad5-nCoV. The intramuscular injection method was 100 ⁇ L injection into the inner thigh, and the nasal immunization method was to anaesthetize the mouse with isoflurane and instill 20 ⁇ L into the nasal cavity. The grouping situation is shown in Table 1.
  • Ad5-nCoV_Sopt mice intramuscularly at day 9 can produce higher serum IgG antibodies, wherein the average value of the high dose group antibody titers of 10 5 or more; IgG antibody levels induced by intramuscular injection of Ad5-nCoV_Sopt sera showed significant dose Depending on the relationship, the higher the dose, the higher the serum IgG antibody level ( Figure 4). From the 9th day after the immunization to the 14th day after the immunization, the serum antibody level further increased, and the serum IgG antibody titers of the high-dose group and the low-dose group were significantly higher on the 14th day after the immunization than on the 9th day ( Figure 5).
  • the serum IgG antibody levels induced by intramuscular injection of the three recombinant adenoviruses Ad5-nCoV_Sopt, Ad5-nCoV_oriSIP-Sopt and Ad5-nCoV_S1opt were analyzed, and the results are shown in Figure 6, where A, the serum IgG antibody drops on day 9 after immunization Degree; B, serum IgG antibody titer on the 14th day after immunization. The results showed that among the three recombinant adenoviruses, the immune response induced by Ad5-nCoV_Sopt was the fastest.
  • mice Thirty 6-8 weeks old SPF female BALB/c mice were randomly divided into three groups, each with 10 mice. Inoculate 5 ⁇ 10 8 VP Ad5-nCoV_Sopt by intramuscular injection (im.) or intranasal immunization (in.) respectively; and inoculate 1 ⁇ 10 7 ifu Ad5 vector as a control with the same immunization method. Each immunization of the control group 5 in each way.
  • the intramuscular injection method was 100 ⁇ L injection into the inner thigh, and the nasal immunization method was to anaesthetize the mouse with isoflurane and instill 20 ⁇ L into the nasal cavity.
  • the experimental grouping situation is shown in Table 2.
  • mice were sacrificed 14 days after immunization, splenic lymphocytes were isolated, and cultured with S protein overlapping peptide library for 6 hours, while adding protein secretion blocking agent to block cytokine secretion. After 6 hours, Fc receptors were blocked, dead cells and cell surface molecular markers were stained. After the cells were fixed and perforated, intracellular cytokines were stained. Cell surface markers include CD3, CD4, CD8 and CD107a molecules, and intracellular cytokines include IFN ⁇ , TNF ⁇ and IL2. A flow cytometer (BD FACS Canto TM ) was used to analyze the levels of CD4 + T cells and CD8 + T cells that express IFN ⁇ , TNF ⁇ , IL2 and CD107a after stimulation with specific peptides.
  • BD FACS Canto TM was used to analyze the levels of CD4 + T cells and CD8 + T cells that express IFN ⁇ , TNF ⁇ , IL2 and CD107a after stimulation with specific peptides.
  • FIG. 8 The CD8 + T cell and CD4 + T cell immune responses induced by Ad5-nCoV_Sopt are shown in Figure 8 and Figure 9.
  • the representative results are shown in Figures 10 to 12, where Figure 10 is a representative diagram of the cellular immune response in the intramuscular injection group.
  • Figure 11 is a representative diagram of cellular immune response in the nasal drip immunization group, and
  • Figure 12 is a representative diagram of cellular immune response in the control group.
  • the results show that whether it is intramuscular injection or intranasal immunization, Ad5-nCoV_Sopt can induce obvious cellular immune response in its immunized mice.
  • mice undergoing cellular immune response testing lung washes were collected at the time of execution, and ELISA method was used to detect the levels of IgG and IgA antibodies against the new coronavirus S1 protein in the lung washes.
  • the experimental groupings are shown in Table 2, and the test results are shown in Figure 13 (****, P ⁇ 0.0001), where A is the level of IgG antibody in the lung wash; B, the level of IgA antibody in the lung wash.
  • the results showed that 14 days after Ad5-nCoV_Sopt immunization, mice immunized with intranasal drip could induce higher levels of lung wash IgG antibody and IgA antibody titers, which were significantly higher than those of the intramuscular injection group and the control group. In the intramuscular injection group, only low levels of lung wash IgG antibody titers could be detected, but no lung wash IgA antibodies were detected.
  • Ad5-nCoV_Sopt, Ad5-nCoV_oriSIP-Sopt and Ad5-nCoV_S1opt three recombinant adenoviruses all have good immunogenicity and can induce mice to produce high levels of serum IgG antibodies. Among them, the serum IgG antibody titer induced by Ad5-nCoV_Sopt is the fastest, and its immunogenicity is the best. Ad5-nCoV_Sopt was selected as the best candidate for the recombinant novel coronavirus vaccine, and it was recorded as Ad5-nCoV.
  • Fifty-six SPF guinea pigs weighing 200 to 250 grams were randomly divided into 4 groups, each with 14 guinea pigs, half male and half female.
  • Guinea pigs were immunized with Ad5-nCoV according to the grouping situation shown in Table 3.
  • the immunization method is the intramuscular injection of 200 ⁇ L into the inner thigh.
  • Guinea pigs collect blood at a specific time point after immunization, separate the serum, and use the ELISA method to detect the IgG antibody titer against the new coronavirus S protein in the serum.
  • the test results are shown in Figure 14 (ns, P ⁇ 0.05; *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001).
  • the results showed that 14 days after Ad5-nCoV immunized guinea pigs, high levels of serum IgG antibody titers were detected. There was no significant difference in antibody titers between the middle-dose group and the high-dose group. The low-dose group had a lower serum IgG antibody level, which was significantly lower than the high-dose group and the low-dose group.
  • the test results show that Ad5-nCoV has good immunogenicity in the guinea pig model.
  • the SARS-CoV-2 (SARS-CoV-2/WH-09/human/2020/CHN) live virus challenge test was carried out in a biosafety level 3 laboratory.
  • mice in the model group lost their body weight after infection, with the highest average reduction percentage being 3.36%.
  • the body weight of the mice in the high-dose group increased to a certain extent on the third day after infection, with an average increase percentage of 2.55%.
  • the average weight loss of mice in the low-dose group was 4.72% on the third day after infection. It showed that the mice in the high-dose group had no obvious symptoms after infection (Figure 15).
  • the lung tissue viral load test result of the model group of mice 3 days after infection was 10 6.18 copies/mL.
  • the lung tissue viral load of mice in the high-dose group was 10 3.11 copies/mL at 3 days after infection, which was significantly lower than that of the model group (p ⁇ 0.001).
  • the lung tissue viral load of mice in the low-dose group was 10 3.90 copies/mL at 3 days after infection, which was significantly lower than that of the model group (p ⁇ 0.001). It showed that the viral load of lung tissue decreased by 3.07lg after high-dose Ad5-nCoV vaccination, and the viral load of lung tissue decreased by 2.28lg after low-dose Ad5-nCoV vaccination. The results of the study suggest that Ad5-nCoV has a significant protective effect on infected mice.
  • the present invention provides a novel coronavirus vaccine using human replication-deficient adenovirus type 5 as a carrier, a preparation method and application in preparing vaccine medicines.
  • the novel coronavirus vaccine provided by the invention is easy to industrially produce and has industrial practicability.

Abstract

提供一种以人5型复制缺陷腺病毒为载体的新型冠状病毒疫苗。该疫苗以E1、E3联合缺失的复制缺陷型人5型腺病毒为载体，以整合腺病毒E1基因的HEK293细胞为包装细胞系，携带的保护性抗原基因是经过优化设计的2019新型冠状病毒（SARS-CoV-2）S蛋白基因（Ad5-nCoV）。该疫苗在小鼠和豚鼠模型上均具有良好的免疫原性，能在短时间内诱导机体产生强烈的细胞及体液免疫反应。hACE2转基因小鼠上的保护效果研究显示，单次免疫Ad5-nCoV14天后能够明显降低肺组织内部的病毒载量，说明该疫苗对2019新型冠状病毒具有良好的免疫保护效果。

Description

一种以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗 技术领域
本发明涉及一种重组新型冠状病毒疫苗，目的在于预防新型冠状病毒疫情。本发明属于生物工程技术领域。
背景技术
2019新型冠状病毒，即SARS-CoV-2(也被称为2019-nCoV)，2020年1月12日被世界卫生组织命名。它是以前从未在人体中发现的冠状病毒新毒株。该病毒为第七个可以感染人的冠状病毒(CoV)。人感染2019-nCoV的潜伏期一般为1～14天，感染2019-nCoV后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。截止到2020年3月17日，我国报告确诊病例81116人，死亡病例3231例；世界其他国家报告病例98486例，累计死亡3848例，平均死亡率3.94％。
新型冠状病毒属于β属冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60～140nm。病毒全基因组系统发育分析(29,903个核苷酸)表明该病毒与一组以前在中国蝙蝠身上发现过的SARS样冠状病毒(genus Betacoronavirus,subgenus Sarbecovirus)关系最密切(89.1％的核苷酸相似性)。在S基因进化树中，2019-nCoV与蝙蝠冠状病毒bat-SL-CoVZC45存在最为密切的亲缘关系，氨基酸一致性为82.3％，而与SARS-CoV的氨基酸一致性大约为77.2％。2019-nCoV进入人体细胞时结合的受体与SARS-CoV的相同，均为血管紧张素转换酶2(ACE2)。
技术问题
冠状病毒是一个大型的病毒家族，迄今引起人群感染暴发主要是SARS-CoV、MERS-CoV以及SARS-CoV-2。有关SARS-CoV-2的结构、功能、感染关键靶点、作用机制及疫苗开发的报道并不多，很多研究主要集中在 SARS-CoV、MERS-CoV疫苗上，主要研发类型有病毒载体疫苗、核酸疫苗、亚单位疫苗、病毒样颗粒疫苗、灭活疫苗及减毒活疫苗等。重组冠状病毒疫苗的靶抗原一般都是采用全长S蛋白和截短型S蛋白(S1和RBD)形式。关于新型冠状病毒疫苗在研的种类包括腺病毒载体疫苗、mRNA疫苗、DNA疫苗、重组蛋白疫苗和灭活疫苗，研究结果还未见报道。本发明的目的就是提供一种重组新型冠状病毒疫苗。
技术解决方案
基于上述目的，本发明首先提供了一种用于编码新型冠状病毒疫苗的S蛋白基因优化的多核苷酸，所述的多核苷酸的序列如SEQ ID NO:1所示。所述多核苷酸以E1、E3联合缺失的复制缺陷型人5型腺病毒为载体，以整合腺病毒E1基因的HEK293细胞为包装细胞系，经包装获得重组腺病毒载体新型冠状病毒疫苗。
本发明还提供了含有上述多核苷酸的载体。
在一个优选的实施方案中，所述载体为pDC316。
本发明还提供了一种能够表达上述多核苷酸的人复制缺陷重组腺病毒。
在一个优选的实施方案中，所述重组腺病毒来源于AdMax腺病毒系统。
本发明还提供了上述重组腺病毒在制备预防新型冠状病毒肺炎疫苗中的应用。
在一个优选的实施方案中，上述应用中，所述重组腺病毒被制备为注射剂、滴鼻剂或喷雾剂。
在一个更为优选的实施方案中，所述重组腺病毒被制备为肌肉注射剂。
最后，本发明提供了一种制备上述的可表达新型冠状病毒S蛋白的重组腺病毒的方法，所述方法包括以下步骤：
(1)构建含有编码2019新型冠状病毒S蛋白的多核苷酸的穿梭质粒载体；
(2)将步骤(1)所述穿梭质粒载体与骨架质粒一起转染入宿主细胞；
(3)培养步骤(2)所述宿主细胞；
(4)收获从步骤(3)所述细胞中释放的人复制缺陷重组腺病毒；
(5)对步聚(4)中的重组腺病毒进行扩大培养；
(6)对步聚(5)中的培养产物进行纯化。
优选的，步骤(1)所述穿梭质粒载体为pDC316。
优选的，步骤(2)所述骨架质粒为pBHGlox_E1,3Cre，两种质粒均属于AdMax腺病毒系统，共同用于在宿主细胞中进行含有编码新型冠状病毒S蛋白的多核苷酸的重组腺病毒包装。
优选的，步骤(3)所述细胞为HEK293细胞。
优选的，步骤(5)所述扩大培养方法为悬浮培养。
优选的，步骤(6)所述纯化方法为Source 30 Q层析法。
技术效果
本发明提供的可表达新型冠状病毒S蛋白的重组腺病毒作为新型冠状病毒疫苗(Ad5-nCoV)，其在小鼠和豚鼠模型上均具有良好的免疫原性，能在短时间内诱导机体产生强烈的细胞及体液免疫反应。在hACE2转基因小鼠上的保护效果研究显示，单次免疫Ad5-nCoV14天后能够明显降低肺组织内的病毒载量。说明该疫苗对2019新型冠状病毒具有良好的免疫保护效果。该疫苗制备方法快速简便，可在短期内实现大规模生产用于应对突发疫情。
附图说明
图1.穿梭质粒pDC316-nCoV_Sopt图谱；
图2.S蛋白细胞内表达Western blot鉴定图谱；
图3.重组新型冠状病毒疫苗候选株原代毒种目标抗原表达Western blot鉴定图谱；
图4.小鼠肌肉注射免疫Ad5-nCoV第9天血清IgG抗体水平比较图；
图5.小鼠肌肉注射免疫Ad5-nCoV第9天和14天血清IgG抗体水平比较图；
图6.小鼠肌肉注射免疫不同构建重组腺病毒血清IgG抗体水平比较图；
图7.小鼠免疫重组腺病毒第14天不同免疫方式诱导的血清抗体水平比较图；
图8.Ad5-nCoV诱导产生的CD8 +T细胞免疫反应比较图；
图9.Ad5-nCoV诱导产生的CD4 +T细胞免疫反应比较图；
图10.肌肉注射组细胞免疫反应代表图；
图11.滴鼻免疫组细胞免疫反应代表图；
图12.对照组细胞免疫反应代表图；
图13.Ad5-nCoV免疫后14天肺洗液抗体水平比较图；
图14.Ad5-nCoV豚鼠模型免疫原性检测比较图；
图15.SARS-CoV-2攻毒后小鼠体重下降百分比比较图；
图16.SARS-CoV-2攻毒后小鼠肺组织病毒载量比较图。
本发明的实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1.以人复制缺陷腺病毒为载体的重组新型冠状病毒疫苗的制备
1.S蛋白基因优化及合成
重组新型冠状病毒疫苗的目标抗原为新型冠状病毒毒株(Genebank编号：NC_045512.2)的S蛋白。通过对S蛋白基因进行优化，提高S蛋白的表达水平，从而提高了疫苗的免疫原性。
首先，使用Upgene软件(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.)进行密码子优化，将S蛋白基因的大部分稀有密码子更改为高使用频率密码子。其次，考虑到软件优化可能机械地将密码子更改为最高使用频率的密码子，受到tRNA使用效率、mRNA二级结构等影响，蛋白翻译效率可能并不会显著提升。在此情况下，我们采用人工分析替换部分高频和低频密码子的方法，同时将高频密码子和低频密码子均匀分布于S蛋白基因。同时考虑到提高mRNA中GC含量，有助于增强mRNA的稳定性，我们适当地提高了S蛋白基因的GC含量，并将G、C核苷酸在整个GP基因中尽可能均衡分布。再次，我们将S蛋白的原始信号肽(1aa-12aa)更改为组织纤溶酶原激活物信号肽tPA，以进一步提升S蛋白的表达水平。
基因优化前，新型冠状病毒S蛋白基因的稀有密码子(使用频率<70％)的含量为34％，高使用频率密码子(使用频率>90％)的含量为23％，GC含量为36％。基因优化后，稀有密码子含量下降至3％，高频率密码子含量提升 至81％，GC含量提升至58％，与原始S蛋白基因序列的同源性为70.4％。
S蛋白基因优化之后，在翻译起始密码子前面加入Kozak序列，并在整个序列上游插入酶切位点EcoRI，下游插入酶切位点HindIII，对基因序列进行合成。与此同时，作为对照，将S蛋白基因的原始序列进行合成。S蛋白基因优化序列(酶切位点为EcoRI和HindIII)见SEQ ID NO:1，S蛋白原始基因序列(酶切位点为SmaI和SalI)见SEQ ID NO:2。
2.载体构建和S蛋白体外表达鉴定
2.1载体构建
以上合成的基因序列，分别用HindIII和SalI，或SmaI和SalI进行双酶切，回收目的基因片段，将其连接至AdMax腺病毒系统(加拿大Microbix Biosystems Inc.)的穿梭质粒pDC316上，转化DH5-α感受态，涂布Amp r LB平板，挑单克隆进行菌落PCR鉴定，并对PCR鉴定为阳性的克隆进行测序验证。S蛋白基因序列未进行优化的质粒记为pDC316-nCoV_S，S蛋白基因优化后的质粒记为pDC316-nCoV_Sopt。pDC316-nCoV_Sopt质粒图谱如图1所示。
同时，使用重叠延伸PCR的方法，扩增原始信号肽S蛋白基因优化序列，连接至pDC316载体，记为pDC316-nCoV_oriSIP-Sopt；扩增tPA信号肽S蛋白基因未优化序列，连接至pDC316载体，记为pDC316-nCoV_tPA-S；tPA信号肽S1蛋白(S1蛋白为S蛋白的12-685位氨基酸的截短型蛋白)基因优化序列(相应的基因优化序列为SEQ ID NO:1的第25bp至2103bp)，连接至pDC316载体，记为pDC316-nCoV_S1opt；以及tPA信号肽S1蛋白基因原始序列，连接至pDC316载体，记为pDC316-nCoV_S1。
2.2S蛋白的体外表达鉴定
将以上构建的6个穿梭质粒和pDC316载体，使用转染试剂TurboFect Transfection Reagent(Thermo Scientific,REF,R0531)转染至HEK293细胞，48小时后收细胞进行WB检测。实验方法如下：
转染：实验前一天，HEK293细胞以8×10 5细胞/孔接种6孔板，于37℃、5％CO 2细胞培养箱中培养过夜。转染前1小时，将培养基换成新鲜的含2％FBS的DMEM培养基，每孔2mL。转染时，每个转染孔取对应质粒2μg，加入到200μL无FBS的DMEM培养基中，混匀，加入转染试剂3μL，轻轻混匀，室温放置15分钟。将质粒和转染试剂混合液轻轻滴加到6孔板中，轻摇混匀。 细胞于37℃、5％CO 2细胞培养箱中培养，5小时后将培养基换成新鲜的含10％FBS的DMEM培养基，48小时后收集细胞，制备样品，进行WB检测。
样品制备：转染48小时后，小心地吸弃培养基，用PBS重悬细胞，500g离心5分钟，弃清。细胞用200μL RIPA缓冲液(Thermo Scientific,Prod#89900，另外添加适量蛋白酶抑制剂和核酸酶)重悬，冰浴15分钟，4℃12000rpm离心5分钟，取上清，加入1/3体积含有200mmol/L DTT的4×SDS-PAGE上样缓冲液，95℃加热5分钟，冻存，用于WB检测。
Western blot检测：使用10孔的4-20％SDS-PAGE梯度胶进行SDS-PAGE，上样量每孔30μL。电泳条件：80V，15min；180V，直到溴酚蓝刚好从凝胶中出来为此。将SDS-PAGE胶上的蛋白质通过电转仪转移到硝酸纤维素膜上，电转条件为300mA，1小时。电转完成后，将硝酸纤维素膜用5％脱脂奶粉封闭1小时，然后以1:2000的稀释度加入抗S蛋白兔多克隆抗体(义翘神州，货号40150-T52)，4℃放置过夜。将膜用WB洗涤液洗涤4次，每次于摇床上摇动5分钟。然后加入以1:10000稀释于5％脱脂奶粉的HRP标记的羊抗兔IgG抗体(CST,7074S)，室温孵育1小时。用WB洗涤液将膜洗涤4次，使用Immobilon TM Western Chemiluminescent HRP Subsrate(MILLIPORE,Cat.No.WBKLS0500)进行化学发光反应，使用化学发光成像仪采集不同曝光时间的图像。
以β-actin作为样品检测内参，结果如图2所示，其中1：转染pDC316-nCoV_S细胞；2：转染pDC316-nCoV_tPA-S细胞；3：转染pDC316-nCoV_oriSIP-Sopt细胞；4：转染pDC316-nCoV_Sopt细胞；5：转染pDC316空载体细胞；6：预染Marker；7：转染pDC316空载体细胞；8：转染pDC316-nCoV_S1细胞；9：转染pDC316-nCoV_S1opt细胞。结果显示没有进行基因优化的序列，无论是S全长基因还是S1基因，其质粒转染的细胞均没有检测到相关蛋白的表达；S蛋白基因优化后，可检测到S蛋白表达；将S蛋白原始信号肽更改为tPA信号肽之后，S蛋白的表达水平进一步提升；同时，S1蛋白基因优化后也可以检测到有明显的S1蛋白的表达。
3.重组腺病毒包装、制备和鉴定
3.1重组腺病毒包装
将以上构建好的载体pDC316-nCoV_Sopt、pDC316-nCoV_oriSIP-Sopt和 pDC316-nCoV_S1opt分别与AdMax腺病毒系统的骨架质粒pBHGlox_E1,3Cre共转染HEK293细胞进行重组腺病毒的包装。过程如下：
a)转染前一天，将HEK293细胞接种于六孔板中，每孔8×10 5个细胞,培养基为MEM+10％FBS，置37℃含5％CO 2细胞培养箱中培养过夜。
b)转染当天换液，用新鲜的含10％FBS的MEM培养基继续培养。待细胞生长至底面积的80-90％时，取骨架质粒(pBHGlox_E1,3Cre)和穿梭质粒，用TurboFect转染试剂(Thermo scientific,REF,R0531)按其所附说明书进行转染。具体步骤为：
(1)每个转染孔取骨架质粒3.2μg，穿梭质粒0.8μg，混和均匀；质粒用400μL Opti-MEM培养基进行稀释。
(2)取6μL TurboFect转染试剂加入到稀释于Opti-MEM培养基的质粒中，轻轻混匀。
(3)将转染试剂和质粒混合物室温放置20min，然后加入到细胞中。
c)转染后第二天，将长满的细胞传代于25cm 2细胞培养瓶中，用含5％FBS的MEM培养基继续培养，每天观察，待细胞长满瓶底时，再传入75cm 2细胞培养瓶中，每天观察细胞出毒迹象。出毒现象为细胞变大变圆，呈葡萄状，并开始出现明显噬斑。待细胞大部分病变并从底部脱落时进行收毒。
d)将出毒的细胞重悬，500g离心10分钟，弃上清，细胞用2mL PBS重悬，先后置于-70℃冰箱和37℃水浴锅中反复冻融三次。12000g离心10分钟，收集含病毒的上清液，弃沉淀。
将表达tPA信号肽S全长蛋白基因优化序列的毒种标记为Ad5-nCoV_Sopt，表达原始信号肽S全长蛋白基因优化序列的毒种标记为Ad5-nCoV_oriSIP-Sopt，表达tPA信号肽S1蛋白基因优化序列的毒种标记为Ad5-nCoV_S1opt。
3.2重组腺病毒鉴定
3.2.1 PCR扩增S及S1全序列及测序鉴定
使用pDC316载体的通用引物，扩增S或S1蛋白的全序列，引物序列如下：
pDC316-F:ACGTGGGTATAAGAGGCG
pDC316-R:CGATGCTAGACGATCCAG
取50μL疫苗候选株毒种液，加入2μL蛋白酶K，50℃消化30min释放病毒基因组，以此为模版扩增S蛋白和S1蛋白基因序列。
PCR扩增条件为：
Figure PCTCN2020096024-appb-000001
反应程序为：
Figure PCTCN2020096024-appb-000002
琼脂糖凝胶电泳结果显示三个毒种均能扩增到单一目的条带，片段大小正确。将目的条带进行胶回收并测序，比对结果表明，测序结果序列完全正确。
3.2.2目标抗原表达鉴定
将不同构建重组腺病毒感染HEK293细胞，48小时后收集细胞进行目标抗原的Western blot检测，三个毒种Ad5-nCoV_Sopt、Ad5-nCoV_oriSIP-Sopt和Ad5-nCoV_S1opt均可检测到目标蛋白的明显表达。结果如图3所示，其中1：预染Marker；2和3：Ad5-nCoV_oriSIP-Sopt感染细胞；4和5：Ad5-nCoV_Sopt；6：Ad5-nCoV_S1opt感染细胞。7和8：空白细胞。
3.2.3重组腺病毒培养
HEK293细胞在37℃、5％CO 2条件下120rpm悬浮培养。毒种接种时，将活度大于95％的细胞稀释至1.0×10 6cells/mL，终体积为300mL。P5代重组腺病毒以MOI 10感染HEK293细胞，37℃，5％CO 2，130rpm摇动培养。每隔24小时取样，检测细胞活度和密度。
毒种接种72小时后，当细胞活度下降到40％以下时，将细胞摇瓶先后置于-70℃冰箱和37℃水浴锅中反复冻融两次。加入Benzonase(20U/mL)，34-36℃水浴酶解2小时。12000g离心10分钟，收集含病毒的上清液，弃沉淀。
3.2.4重组腺病毒纯化
用5×平衡缓冲液调节病毒上清液至电导值为18mS/cm，pH7.5。采用Source 30Q分离纯化腺病毒颗粒，层析柱用A液(20mmol/L Tris+150mmol/L NaCl+2mmol/L MgCl 2pH7.5。)平衡，上样，上样流速为5mL/min；上样结束，用A液平衡至Uv基线，10mL/min,50min,0％B-20％B梯度洗脱，分管收集洗脱峰，最后100％B洗脱，B液为20mmol/L Tris+2mol/L NaCl+2mmol/L MgCl 2 pH7.5。
3.3 Ad5-nCoV的鉴定和滴度测定
3.3.1 PCR扩增目标蛋白基因全序列和测序鉴定
实验方法和过程同3.2.1。琼脂糖凝胶电泳结果显示三个毒种均能扩增到单一目的条带，片段大小正确。将目的条带进行胶回收并测序，比对结果表明，测序结果序列完全正确。
3.3.2感染滴度测定
使用Clontech Adeno-X TM Rapid Titer Kit进行重组腺病毒滴度的测定。操作按试剂盒所附说明书进行，具体方法如下：
a)将HEK293细胞接种于24孔板。细胞密度为5×10 5cells/mL，每孔接种0.5mL，培养基为MEM+10％FBS。
b)使用培养基把将要检测的病毒从10 -2至10 -6进行10倍稀释，制备一系列稀释度的病毒样品，每孔50μL加入到细胞中。
c)细胞于37℃、5％CO2培养箱中培养48小时。
d)吸弃细胞的培养基，让细胞稍微晾干(不要过干)。每孔轻轻加入0.5mL冰甲醇，于-20℃放置10分钟，对细胞进行固定。
e)吸弃甲醇，用PBS+1％BSA将细胞轻轻地洗3次。每孔加入0.25mL抗-Hexon抗体稀释液(1:1000稀释)，37℃孵育1小时。
f)吸弃抗-Hexon抗体，用PBS+1％BSA将细胞轻轻洗3次，每孔加入0.25mL HRP标记的Rat Anti-Mouse Antibody(1:500稀释)，37℃孵育1小时。
g)在吸弃0.25mL HRP标记的Rat Anti-Mouse Antibody之前，将10×DAB底物用1×Stable Peroxidase Buffer稀释成1×DAB工作液，让其达到室温。
h)吸弃Rat Anti-Mouse Antibody稀释液，用PBS+1％BSA将细胞轻轻地洗3次。每孔加入0.25mL DAB工作液，室温放置10分钟。
i)吸弃DAB工作液，用PBS将细胞轻轻地洗2次。
j)于显微镜下对棕色/黑色的阳性细胞进行计数。每孔至少随机计数3个视野，计算其平均阳性细胞数。
k)计算感染滴度(ifu/mL)。公式如下：
感染滴度(ifu/mL)＝视野的阳性细胞数×每孔视野数/(病毒体积(mL)×稀释度)
滴度测定结果显示，纯化的重组腺病毒经浓缩后，感染滴度达1.0×10 10ifu/mL以上。
3.3.3病毒颗粒数测定
将20mmol/L Tris-Cl、2mmol/L EDTA(pH 7.5)溶液和2.0％SDS溶液等体积混合，配置成病毒裂解液。取适当体积待测病毒样品，加入1/19体积病毒裂解液，用移液器反复吹打10次混匀，涡旋1分钟。置于56℃恒温水浴中摇动消化10分钟，12000rpm离心5分钟，取上清，测定260nm和280nm下的OD值。计算腺病毒颗粒数。
病毒颗粒数测定结果显示，纯化的重组腺病毒经浓缩后均达到1.0×10 11VP/mL以上。
实施例2.不同构建重组腺病毒在小鼠模型上的免疫学评价
1. 1.疫苗体液免疫反应检测
100只SPF级雌性BALB/c小鼠(6-8周龄)，随机分成10组，每组10只。根据表1所示分组情况对小鼠进行Ad5-nCoV免疫。肌肉注射方式为后大腿内侧注射100μL，滴鼻免疫方式为将小鼠用异氟烷麻醉，经鼻腔滴入20μL。分组情况如表1所示。
表1.疫苗体液免疫反应检测小鼠分组情况
Figure PCTCN2020096024-appb-000003
Figure PCTCN2020096024-appb-000004
小鼠于免疫后特定时间点采血，分离血清，使用ELISA法检测血清中针对新型冠状病毒S蛋白的IgG抗体滴度。检测结果如图4至图7所示(ns,P≥0.05；*,P<0.05；**,P<0.01；***,P<0.001；****,P<0.0001)。
小鼠肌肉注射Ad5-nCoV_Sopt第9天即可产生较高的血清IgG抗体，其中高剂量组抗体滴度平均值达10 5以上；Ad5-nCoV_Sopt肌肉注射诱导产生的血清IgG抗体水平呈现明显的剂量依赖关系，剂量越高，血清IgG抗体水平越高(图4)。从免疫后第9天至免疫后第14天，血清抗体水平进一步提升，其中高剂量组和低剂量组血清IgG抗体滴度在免疫后第14天显著高于第9天(图5)。
对Ad5-nCoV_Sopt、Ad5-nCoV_oriSIP-Sopt和Ad5-nCoV_S1opt三个重组腺病毒肌肉注射诱导产生的血清IgG抗体水平进行分析，结果如图6所示，其中A，免疫后第9天血清IgG抗体滴度；B，免疫后第14天血清IgG抗体滴度。结果显示，三个重组腺病毒之间，以Ad5-nCoV_Sopt诱导产生的免疫反应最为迅速，免疫后第9天，Ad5-nCoV_Sopt产生的血清IgG抗体水平显著高于Ad5-nCoV_oriSIP-Sopt和Ad5-nCoV_S1opt(图6，A)。免疫后第14天，Ad5-nCoV_oriSIP-Sopt的血清抗体水平有所提升(图6，B)，但是Ad5-nCoV_S1opt诱导产生的血清IgG抗体水平无论在免疫后第9天还是第14天均显著低于另外两个疫苗候选株。结果说明，三个重组腺病毒之间，以Ad5-nCoV_Sopt诱导产生的血清IgG抗体滴度最为快速，其免疫原性最好。
对免疫后第14天肌肉注射或滴鼻免疫Ad5-nCoV_Sopt诱导的血清抗体滴 度进行比较分析，结果如图7所示，无论是高剂量，中剂量还是低剂量组，两种免疫诱导产生的血清IgG抗体水平均无显著性差异。
2. 2.细胞免疫反应检测
30只6-8周龄SPF级雌性BALB/c小鼠，随机分成三组，每组10只。分别经肌肉注射(im.)或滴鼻免疫(in.)接种5×10 8VP的Ad5-nCoV_Sopt；并以同样的免疫方式接种1×10 7ifu的Ad5载体作为对照，对照组每种免疫方式各5只。肌肉注射方式为后大腿内侧注射100μL，滴鼻免疫方式为将小鼠用异氟烷麻醉，经鼻腔滴入20μL。实验分组情况如表2所示。
表2.Ad5-nCoV_Sopt小鼠模型细胞免疫反应检测实验分组情况
Figure PCTCN2020096024-appb-000005
免疫后14天处死小鼠，分离脾淋巴细胞，使用S蛋白重叠肽库刺激培养6小时，同时加入蛋白分泌阻断剂阻断细胞因子分泌。6小时后，阻断Fc受体，对死细胞和细胞表面分子标志物进行染色，细胞经固定和穿孔之后，对细胞内细胞因子进行染色。细胞表面标志物包括CD3、CD4、CD8和CD107a分子，细胞内细胞因子包括IFNγ、TNFα和IL2。使用流式细胞仪(BD FACS Canto TM)分析CD4 +T细胞和CD8 +T细胞经特异性肽刺激后表达IFNγ、TNFα、IL2和CD107a的水平。
Ad5-nCoV_Sopt诱导的CD8 +T细胞和CD4 +T细胞免疫反应如图8和图9所示，代表性结果如图10至图12所示，其中图10为肌肉注射组细胞免疫反应代表图，图11为滴鼻免疫组细胞免疫反应代表图，图12为对照组细胞免疫反应代表图。结果显示，无论是肌肉注射还是滴鼻免疫，Ad5-nCoV_Sopt均可诱导其免疫小鼠产生明显的细胞免疫反应。小鼠免疫后14天，脾细胞经S蛋白重叠肽库刺激后，无论是CD8 +T细胞还是CD4 +T细胞，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。与此同时，相比于滴鼻免疫(in.)，肌肉注射(im.)可诱导产生更加强烈的细胞免疫反应。肌肉注射诱导CD8 +T细胞和CD4 +T细胞产生的IFNγ、TNFα和IL2水平均显著高于滴鼻免疫。
3.肺洗液抗体水平检测
进行细胞免疫反应检测的小鼠，处死时同时采集肺洗液，使用ELISA法检测肺洗液中针对新型冠状病毒S1蛋白的IgG和IgA抗体水平。实验分组如表2所示，检测结果如图13所示(****,P<0.0001)，其中A，肺洗液IgG抗体水平；B，肺洗液IgA抗体水平。结果显示，Ad5-nCoV_Sopt免疫后14天，滴鼻免疫小鼠可诱导产生较高水平的肺洗液IgG抗体和IgA抗体滴度，显著高于肌肉注射组和对照组。而肌肉注射组仅能检测到低水平的肺洗液IgG抗体滴度，未能检测到肺洗液IgA抗体。
4. 4.小鼠模型免疫原性评价小结
Ad5-nCoV_Sopt、Ad5-nCoV_oriSIP-Sopt和Ad5-nCoV_S1opt三个重组腺病毒均具有良好的免疫原性，可诱导小鼠产生高水平的血清IgG抗体。其中以Ad5-nCoV_Sopt诱导产生的血清IgG抗体滴度最为快速，其免疫原性最好。选择Ad5-nCoV_Sopt作为重组新型冠状病毒疫苗最佳候选，将其记为Ad5-nCoV。细胞免疫反应检测结果说明，肌肉注射和滴鼻免疫Ad5-nCoV均可诱导其免疫的小鼠产生特异性细胞免疫反应，其中肌肉注射免疫方式可诱导强烈的特异性细胞免疫反应。肺洗液抗体检测结果说明，滴鼻免疫可诱导产生较高水平的肺洗液IgG和IgA抗体。
实施例3.Ad5-nCoV在豚鼠模型上的免疫学评价
56只SPF级豚鼠，体重200至250克，随机分成4组，每组14只，雌雄各半。根据表3所示分组情况对豚鼠进行Ad5-nCoV免疫。免疫方式为后大腿内侧肌肉注射200μL。
表3.Ad5-nCoV豚鼠免疫原性检测分组情况
Figure PCTCN2020096024-appb-000006
豚鼠于免疫后特定时间点采血，分离血清，使用ELISA法检测血清中针对新型冠状病毒S蛋白的IgG抗体滴度。检测结果如图14所示(ns,P≥0.05；*, P<0.05；**,P<0.01；***,P<0.001；****,P<0.0001)。结果显示，Ad5-nCoV免疫豚鼠14天后，检测到高水平的血清IgG抗体滴度。中剂量组与高剂量组之间抗体滴度无显著性差异，低剂量组血清IgG抗体水平较低，显著低于高剂量组与低剂量组。检测结果表明，Ad5-nCoV在豚鼠模型上具有良好的免疫原性。
实施例4.Ad5-nCoV在hACE2转基因小鼠模型上的保护效果评价
在生物安全3级实验室中进行SARS-CoV-2(SARS-CoV-2/WH-09/human/2020/CHN)活病毒攻毒试验。
实验分为3组(表4)，分别后腿肌肉注射Ad5-nCoV 5×10 9VP或5×10 8VP，注射体积为100μL，或肌肉注射等量体积的PBS。免疫后2周将小鼠转移至生物安全3级实验室，进行SARS-CoV-2攻毒，攻毒方式为滴鼻(10 5TCID 50/只。小鼠攻毒后连续观察3天，记录体重变化。感染后第3天安乐死全部小鼠，检测肺组织病毒载量。
表4.Ad5-nCoV动物保护试验分组情况。
Figure PCTCN2020096024-appb-000007
模型组小鼠感染后出现体重下降，平均下降百分比最高为3.36％。与模型组比较，高剂量组小鼠感染后第3天体重有一定程度升高，平均升高百分比为2.55％。低剂量组小鼠感染后第3天体重平均下降百分比为4.72％。显示给予高剂量组小鼠感染后无明显症状(图15)。在病毒载量方面，如图16所示，模型组小鼠感染后3天肺组织病毒载量检测结果为10 6.18copies/mL。高剂量组小鼠感染后3天肺组织病毒载量为10 3.11copies/mL，显著低于模型组(p<0.001)。低剂量组小鼠感染后3天肺组织病毒载量为10 3.90copies/mL，显著低于与模型组(p<0.001)。显示高剂量Ad5-nCoV接种后肺组织病毒载量下降3.07lg值，低剂量Ad5-nCoV免疫接种后肺组织病毒载量下降2.28lg值。研究结果提示Ad5-nCoV对感染小鼠有明显保护作用。
工业实用性
本发明提供了一种以人5型复制缺陷腺病毒为载体的新型冠状病毒疫苗， 制备方法以及在制备疫苗药物中的应用，本发明提供的新型冠状病毒疫苗易于工业化生产，具有工业实用性。
序列表自由内容
Figure PCTCN2020096024-appb-000008
Figure PCTCN2020096024-appb-000009
Figure PCTCN2020096024-appb-000010
Figure PCTCN2020096024-appb-000011
Figure PCTCN2020096024-appb-000012
Figure PCTCN2020096024-appb-000013

Claims (14)

  1. 一种编码2019新型冠状病毒S蛋白的多核苷酸，其特征在于，所述多核苷酸的序列如SEQ ID NO:1所示。
  2. 一种含有权利要求1所述多核苷酸的载体。
  3. 根据权利要求2所述的载体，其特征在于，所述载体为pDC316。
  4. 一种能够表达权利要求1所述多核苷酸的人复制缺陷重组腺病毒。
  5. 根据权利要求4所述的重组腺病毒，其特征在于，所述重组腺病毒来源于AdMax腺病毒系统。
  6. 权利要求4和5所述重组腺病毒在制备预防2019新型冠状病毒疫苗中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述重组腺病毒被制备为注射剂、滴鼻剂或喷雾剂。
  8. 根据权利要求7所述的应用，其特征在于，所述重组腺病毒被制备为肌肉注射剂。
  9. 一种制备权利要求4或5所述的重组腺病毒的方法，所述方法包括以下步骤：
    (1)构建含有编码2019新型冠状病毒S蛋白的多核苷酸的穿梭质粒载体；
    (2)将步骤(1)所述穿梭质粒载体与骨架质粒一起转染入宿主细胞；
    (3)培养步骤(2)所述宿主细胞；
    (4)收获从步骤(3)所述细胞中释放的人复制缺陷重组腺病毒；
    (5)对步聚(4)中的重组腺病毒进行扩大培养；
    (6)对步聚(5)中的培养产物进行纯化。
  10. 根据权利要求9所述的方法，其特征在于，步骤(1)所述载体为pDC316。
  11. 根据权利要求9所述的方法，其特征在于，步骤(2)所述骨架质粒为pBHGlox_E1,3Cre。
  12. 根据权利要求9所述的方法，其特征在于，步骤(3)所述细胞为HEK293细胞。
  13. 根据权利要求9所述的方法，其特征在于，步骤(5)所述扩大培养方法为悬浮培养。
  14. 根据权利要求9所述的方法，其特征在于，步骤(6)所述纯化方法为Source 30Q层析法。
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