WO2021000968A2 - 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗 - Google Patents

一种用于预防SARS-CoV-2感染的腺病毒载体疫苗 Download PDF

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WO2021000968A2
WO2021000968A2 PCT/CN2020/110085 CN2020110085W WO2021000968A2 WO 2021000968 A2 WO2021000968 A2 WO 2021000968A2 CN 2020110085 W CN2020110085 W CN 2020110085W WO 2021000968 A2 WO2021000968 A2 WO 2021000968A2
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vaccine
vector
cov
immunization
rhesus monkeys
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WO2021000968A3 (zh
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陈凌
关素华
杨臣臣
刘晓琳
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广州恩宝生物医药科技有限公司
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Priority claimed from CN202010110070.8A external-priority patent/CN110951756B/zh
Priority claimed from CN202010145657.2A external-priority patent/CN110974950B/zh
Application filed by 广州恩宝生物医药科技有限公司 filed Critical 广州恩宝生物医药科技有限公司
Priority to EP20829299.5A priority Critical patent/EP3804751A4/en
Priority to US17/260,820 priority patent/US20210283244A1/en
Priority to PH12021550029A priority patent/PH12021550029A1/en
Publication of WO2021000968A2 publication Critical patent/WO2021000968A2/zh
Publication of WO2021000968A3 publication Critical patent/WO2021000968A3/zh

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Definitions

  • the invention relates to an adenovirus vector vaccine for preventing SARS-CoV-2 infection.
  • the incubation period of the new coronavirus SARS-CoV-2 can be as long as 24 days and is highly contagious. Unlike the SARS virus that causes SARS, some cases are infectious during the incubation period, and some carriers of the virus do not show any obvious symptoms. This makes the prevention and control of the epidemic more difficult. As of July 20, 2020, the total number of confirmed cases worldwide is close to 15 million, and the death toll exceeds 600,000. So far, no specific drugs for COVID-19 have been found. To control the epidemic, most countries implemented isolation measures and closed non-essential facilities, causing huge economic losses. Therefore, the rapid development of a preventive vaccine that can improve the level of herd immunity and block the spread of the virus has become the most urgent and major need.
  • Adenovirus is a commonly used vector in vaccine development and gene therapy, and is widely used in the field of biomedicine. Compared with other vectors (such as bacteria or other viruses), adenovirus has low toxicity, and infection with adenovirus only causes mild cold symptoms.
  • the Ad5 empty vector virus is a replication-defective Ad5 vector lacking genes in the E1 and E3 regions and does not carry any foreign genes. The safety of gene therapy products using Ad5 as a carrier has been confirmed in multiple clinical trials.
  • SARS-CoV-2 The full sequence of SARS-CoV-2 is shown in NC_045512.2, and the 21563..25384 nucleic acid of this sequence is the coding sequence of Spike protein (S).
  • S protein is a major structural protein of viral particles, which plays an important role in mediating the binding of viral particles to host cell receptors and inducing neutralizing antibodies. Therefore, for vaccines with S protein as antigen, including nucleic acid vaccines, subunit vaccines and viral vector vaccines, the expression level and protein structure of the S protein determine the effectiveness of the vaccine.
  • the experiment surface shows that the expression level of the natural spike protein S gene of SARS-CoV-2 in humans is low. If the original S codon is used as an antigen, it is not enough to produce enough antigen, and the vaccine may be ineffective or The titer is very low, not enough to resist virus infection.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide an optimized S protein nucleotide sequence.
  • the nucleotide sequence is in 1) DNA plasmid; 2) After being integrated into adenovirus vector, it can express and produce more S protein after transfection into human cell line, which is expected to be used as antigen gene of nucleic acid vaccine or recombinant virus vaccine for prevention SARS-CoV-2 infection.
  • the first aspect of the present invention provides:
  • a vaccine for preventing SARS-CoV-2 infection comprising:
  • nucleotide sequence that has at least 80%, 85%, 90%, 95%, 100% homology with the nucleotide shown in SEQ ID NO:1.
  • the nucleotide sequence can express a protein in a human cell or in a human body.
  • the protein can be in the human body:
  • the induced immune response includes antibody and cell-mediated immune response
  • the vaccine further includes a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.
  • the vaccine also includes at least one drug that has a therapeutic effect on COVID-19.
  • the second aspect of the present invention provides:
  • An expression vector containing the nucleotide sequence described in the first aspect of the present invention is an expression vector containing the nucleotide sequence described in the first aspect of the present invention.
  • the transcription direction of the nucleotide sequence described in the first aspect of the present invention is opposite to the transcription direction of other genes of the vector. This can further ensure that the expressed protein has higher purity.
  • the vector is a DNA plasmid or RNA expression plasmid or viral vector.
  • the viral vector is an adenovirus vector.
  • the adenovirus vector is a replication-deficient Ad5 vector.
  • the third aspect of the present invention provides:
  • the application of the vaccine according to the first aspect of the present invention includes:
  • the fourth aspect of the present invention provides:
  • a method for preventing or treating COVID-19 includes administering a preventive or therapeutic amount of vaccine to a human, and the vaccine is as described in the first aspect of the present invention.
  • the vaccines of some examples of the present invention contain S protein nucleotide sequences that are easy to express in human cells and can induce the production of more S protein, and are expected to be used as a recombinant virus vaccine to prevent SARS-CoV-2 infection.
  • Some examples of the present invention have better safety.
  • the animal experiment results of some examples of the present invention show that high titer antibodies and cellular immunity can be produced after immunizing mice and macaques.
  • Rhesus monkeys immunized with a single intramuscular injection or nasal spray/oral drip of the vaccine can be challenged 30 days after immunization, which can completely resist viral infection.
  • Rhesus monkeys that received a single 1/10 low-dose (1 ⁇ 10 10 VP/mouse) vaccine intramuscularly were challenged 8 weeks after immunization, which can also completely resist viral infection. After the vaccine group rhesus monkeys were challenged, no lung damage caused by ADE or virus was observed.
  • the present invention in addition to stimulating the body's humoral immunity, the present invention also produces cellular immunity, which may further improve the protection of the body.
  • nasal and oral immunization can also obtain good protection, suggesting the feasibility of mucosal immunization and non-injection immunization.
  • Figure 1 is the detection result of the expression of S protein after the plasmid containing the codon-optimized S gene nucleotide sequence is transfected into human cells.
  • Figure 2 is an experimental result of animal immunogenicity evaluation, showing that Ad5-NB2 can induce mice to produce antibodies.
  • Figure 3 shows the detection results of binding antibodies in serum at different times after immunization of rhesus monkeys
  • a detection results of binding antibodies in serum at different times after immunization of rhesus monkeys
  • b detection results of binding antibodies in serum of rhesus monkeys 18 days after immunization.
  • Figure 4 shows the titers of neutralizing antibodies against the new coronavirus live virus in serum after immunization of rhesus monkeys.
  • Figure 5 shows the ELISpot test results of PBMCs in peripheral blood of rhesus monkeys 18 days after immunization.
  • Figure 6 is the determination of throat swab virus genome copy number after rhesus monkey challenge, a: the change of virus genome copy number after challenge in the control group b: the change of virus genome copy number after intramuscular injection of high dose group c: intramuscular injection of low dose challenge Changes in virus genome copy number after virus d: Changes in virus genome copy number after nasal spray + mouth challenge.
  • Figure 7 shows the peak viral load in throat swabs from vaccinated and non-vaccinated rhesus monkeys.
  • Each open circle represents the peak viral load of a rhesus monkey.
  • Diamonds are rhesus monkeys inoculated by high-dose intramuscular injection; squares are rhesus monkeys inoculated by nasal spray + mouth drop; triangles are rhesus monkeys inoculated by low-dose intramuscular injection; black circles are non-vaccinated macaques.
  • Figure 8 is the determination of the copy number of the viral genome in different parts of the macaque after challenge.
  • Figure 9 shows the lung pathological results of rhesus monkeys after challenge.
  • a Pathological section of the lungs of rhesus monkeys in the control group after challenge (HE staining)
  • b Pathological sections of the lungs of rhesus monkeys in the control group (HE staining)
  • c the lungs of the high dose group (1 ⁇ 10 11 VP) after intramuscular injection Pathological section (HE staining)
  • d nasal drip + oral drip combined immunization group (5 ⁇ 10 10 vp/route) and lung pathological section after challenge (HE staining)
  • e intramuscular low-dose group (1 ⁇ 10 10 VP) ) Pathological section of lung after challenge (HE staining).
  • Figure 10 shows the changes of anti-SARS-CoV-2 neutralizing antibodies before and after rhesus challenge.
  • a Changes in anti-SARS-CoV-2 neutralizing antibodies before and after challenge in the high-dose intramuscular injection group
  • b Changes in anti-SARS-CoV-2 neutralizing antibodies before and after challenge in the nose/dose group
  • c Low-dose intramuscular injection group Changes in neutralizing antibodies against SARS-CoV-2 before and after
  • d Changes in neutralizing antibodies against SARS-CoV-2 before and after challenge in the control group.
  • Figure 11 shows the changes of anti-Ad5 neutralizing antibodies before and after immunization in rhesus monkeys.
  • a The changes of anti-Ad5 neutralizing antibodies in the high-dose intramuscular injection group before and after immunization.
  • b Changes in anti-Ad5 neutralizing antibodies before and after immunization in the low-dose intramuscular injection group
  • c Changes in anti-Ad5 neutralizing antibodies before and after immunization in the nose/dose group
  • d Changes in anti-Ad5 neutralizing antibodies before and after immunization in the control group.
  • the amino acid sequence of the spike protein (S) of SARS-CoV-2 is shown in YP_009724390.1 and is denoted as NB1.
  • the mRNA precursors transcribed by eukaryotic cells can produce different mRNA splicing isoforms through different splicing methods (choose different splicing site combinations), and finally lead to different proteins produced by the same gene sequence. This is very detrimental to protein expression.
  • the inventors optimized the wild-type natural nucleotide sequence with codons and removed potential variable splicing sites based on their own technology to ensure the uniqueness of protein expression and reduce the difficulty of subsequent protein purification.
  • the optimized nucleotide sequence is denoted as NB2, and its specific sequence is shown in SEQ ID NO: 1:
  • NB4 the synthesized sequence was named NB4, as shown in SEQ ID NO.: 2, the sequence identity of NB4 and NB2 was 85.93%.
  • NB1, NB2 and NB4 were used as templates, NB1-F and NB1-R were used as primers for PCR amplification to obtain NB1 fragments, NB2-F and NB2-R were used as primers for PCR amplification to obtain NB2 fragments, with NB4-F and NB4 -R is the primer PCR amplified to obtain the NB4 fragment, and then each CMV-R and BGH-F are used as primers, and the company’s existing pGA1-EGFP plasmid is used as the template.
  • the same Source recombinase (Exnase) was recombined with NB1, NB2 and NB4 fragments in vitro to obtain pGA1-NB1, pGA1-NB2 and pGA1-NB4.
  • the pGA1-NB2 was linearized with Bstz17I and SgarAI, and the linearized pAd5 ⁇ E1 ⁇ E3 (ie Ad5 empty vector) was digested with BJ5183 competence and the only restriction site corresponding to delete the E1 region for homologous recombination to construct pAd5-NB2 Vaccine carrier.
  • the primer sequence for amplifying NB1 is the primer sequence for amplifying NB1:
  • NB1-F GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTTGTTTTTCTTGT (SEQ ID NO.: 3)
  • NB1-R AGAATAGGGCCCTCTAGACTAGTTTATGTGTAATGTAATTTG (SEQ ID NO.: 4)
  • the primer sequence for amplifying NB2 is the primer sequence for amplifying NB2
  • NB2-F GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTCGTGTTTCTGGT (SEQ ID NO.: 5)
  • NB2-R AGAATAGGGCCCTCTAGACTAGTTTATCAGGTGTAGTGCAGCTTC (SEQ ID NO.: 6)
  • the primer sequence for amplifying NB4 is the primer sequence for amplifying NB4
  • NB4-F GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTTGTCTTCCTGGT (SEQ ID NO.: 7)
  • NB4-R AGAATAGGGCCCTCTAGACTAGTTTAGGTGTAGTGCAGCTTC (SEQ ID NO.: 8)
  • the primer sequence for amplifying pGA1 is the primer sequence for amplifying pGA1:
  • BGH-F TCTAGAGGGCCCTATTCTATAGTGTC (SEQ ID NO.: 9)
  • CMV-R GGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACGCTAGAGTCCGG (SEQ ID NO.: 10)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C 2min; cycles 30; 72°C, 5min.
  • pAd5-NB2 was linearized with AsiSI, recovered by ethanol precipitation, and transfected into 293 cells by cationic liposome transfection method. 8 hours after transfection, add 2 ml of DMEM medium containing 5% fetal bovine serum, Incubate for 7-10 days, observe cell pathology;
  • pGA-NB1, pGA-NB2 and pGA-NB4 were transfected for 48 hours, and then the cells were collected.
  • HEK293 cells were infected with Ad5-NB2 and Ad5 empty vector viruses, and the cells were collected 24h later.
  • the above four samples were processed according to the conventional Western Blot method, and protein detection was performed ( Figure 1).
  • mice aged 6-8 weeks were divided into 5 groups with 5 mice in each group; on day 0, the dose of Ad5-NB2 was injected intramuscularly: 5 ⁇ 109vp/mouse, and intramuscular injection and nasal drip were used. On the 8th day, blood was taken from the orbit and the serum was separated. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of antibodies in serum with the S protein of the new coronavirus as the antigen. The specific operation is:
  • the macaques are from Guangdong Landao Biotechnology Co., Ltd.
  • the age of the vaccinated macaques is 6 to 14 years old.
  • the immunization dose of each rhesus monkey is 5 ⁇ 10 10 VP.
  • Vaccines were prepared with Ad5-NB2 strain to immunize rhesus monkeys, and blood was collected 12 days, 18 days, and 24 days after immunization.
  • the antibody binding titer was determined by ELISA, and the neutralizing antibody titer was tested for true virus.
  • the separated peripheral blood was tested by ELISpot method. Cellular immune response.
  • the serum of 4 rhesus monkeys injected with 1 ⁇ 10 11 VP and 3 rhesus monkeys injected with 1 ⁇ 10 10 VP could detect significant S-specific IgG.
  • S-specific IgG can be detected in the serum of all immunized rhesus monkeys on 24 days. In rhesus monkeys that received low or high dose immunization, S-specific IgG continued to increase over time (Figure 3a).
  • the neutralizing antibody titer in the serum sample was determined by using the neutralization reaction between the new coronavirus live virus (2019-nCoV-WIV04 strain) and the serum antibody.
  • the obtained animal serum samples were heat-inactivated at 56°C for 30 minutes, diluted to 1:50, 1:150, 1:450, 1:1350, 1:4050 and 1:12150, and the same amount of live virus was added at 37°C, After culturing for 3 days under 5% CO 2 condition, the cells were fixed with 4% formaldehyde and then stained with crystal violet. Count the number of plaques and determine the neutralizing antibody titer (EC 50 ).
  • Ad5-NB2 can also cause cellular immune responses in non-human primates (NHPs)
  • PBMCs peripheral blood mononuclear cells
  • the cellular immune response mainly targets the S1 area, similar to the phenomenon observed in mice.
  • This result shows that intramuscular injection of vaccine can cause systemic cellular immune response to S protein, especially to S1 area, and the systemic cellular immune response caused by vaccine mucosal immunization is weaker.
  • the challenge test was conducted in the P4 laboratory of Wuhan Institute of Virology, Chinese Academy of Sciences. 1 ⁇ 10 11 VP immunization group (intramuscular injection group, nasal/oral drip immunization group) 30 days after immunization, 1 ⁇ 10 10 VP low-dose immunization group (intramuscular injection group) 8 weeks after immunization, intratracheal inoculation 2 ⁇ 10 4 TCID50 SARS-CoV-2 (2019-nCoV-WIV04) for challenge test. Due to space limitations in the P4 laboratory, only 3 high-dose group of intramuscularly injected monkeys, 3 high-dose nasal drops + oral instillation immunized monkeys and 3 low-dose intramuscular immunized monkeys were randomly selected for challenge experiments.
  • the viral load based on the area under the curve (AUC).
  • AUC area under the curve
  • the plaque reduction neutralization test was used to compare serum neutralizing antibody titers before and after challenge.
  • the serum neutralizing antibody titers before the challenge were 1:636, 1:389, and 1:784, respectively; on the 7th day after the challenge, the neutralizing antibody titers were 1:1225, 1:518 and 1:1350, indicating that there is no challenge or the enhancement effect is weak (Figure 10a).
  • the serum neutralizing antibody titers before and on the 7th day after the challenge were relatively low, and there was no significant change.
  • the serum neutralizing antibody titers before the challenge were 1:164, 1:137, and 1:150, respectively; on the 7th day after the challenge, the neutralizing antibody titers were 1:193, 1:168, and 1:130 ( Figure 10b).
  • This replication-deficient type 5 adenovirus vector optimizes the design of S gene NB2, which can significantly increase antigen expression, and can produce high-titer antibodies and cellular immunity after immunizing mice and macaques.
  • Rhesus monkeys immunized with a single intramuscular injection or nasal spray/oral drip of the vaccine can be challenged 30 days after immunization, which can completely resist viral infection.
  • Rhesus monkeys that received a single 1/10 low-dose (1 ⁇ 10 10 VP/mouse) vaccine intramuscularly were challenged 8 weeks after immunization, which can also completely resist viral infection. After the vaccine group rhesus monkeys were challenged, no lung damage caused by ADE or virus was observed.

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Abstract

本发明公开了一种用于预防SARS-CoV-2感染的腺病毒载体疫苗，所述疫苗包括如SEQ ID NO：1所示的核苷酸序列。本发明一些实例的疫苗，其含有的S蛋白核苷酸序列易于在人体细胞表达，可以诱导产生更多的S蛋白，有望作为一种重组病毒疫苗，用于预防SARS-CoV-2感染。本发明的一些实例，具有较好的安全性。

Description

一种用于预防SARS-CoV-2感染的腺病毒载体疫苗 技术领域
本发明涉及一种用于预防SARS-CoV-2感染的腺病毒载体疫苗。
背景技术
新型冠状病毒SARS-CoV-2的潜伏期可长达24天，传染性强，与引发非典的SARS病毒不同，部分病例潜伏期具有传染性，另有一些病毒携带者没有表现出任何明显症状。这对疫情的防控增加了难度。截止2020年7月20日，全球累计确认病例数接近1500万，死亡人数超过60万。迄今尚未发现COVID-19的特效药物。为控制疫情，多数国家实行隔离措施，关闭非必要的设施，造成了巨大的经济损失。因此，快速研制出能够提升群体免疫水平并阻断病毒传播的预防疫苗已成为当前最为紧迫的重大需求。
针对新型冠状病毒，目前国内外尚无明确验证的特效抗病毒药物和预防性疫苗。因此做好预防，阻断病毒的传播是控制疫情的关键。而疫苗的应用已在消除多种传染病中发挥了不可替代的作用。已公布的十几个SARS-CoV-2病毒基因组比对的结果显示病毒之间的差异非常小，目前还没发现发生变异。因此，如果SARS-CoV-2疫苗研发成功，必将能在很大程度上抑制新疫情的暴发。
腺病毒是疫苗研发与基因治疗中常用的载体，被广泛应用于生物医学领域。与其它载体(如细菌或其它病毒)相比，腺病毒的毒性低，感染腺病毒只引起轻微的感冒症状。Ad5空载体病毒为缺失E1和E3区基因的复制缺陷型Ad5载体，不携带任何外源基因。以Ad5为载体的基因治疗产品已在多个临床试验上证实了其安全性。
SARS-CoV-2的全序列如NC_045512.2所示，该序列的21563..25384核酸为刺突蛋白(Spike protein，S)的编码序列。S蛋白是病毒粒子的一种主要结构蛋白，在介导病毒粒子与宿主细胞受体的结合以及诱导中和抗体中起重要作用。因此，以S蛋白为抗原的疫苗，包括核酸疫苗，亚单位疫苗和病毒载体疫苗，其S蛋白的表达水平、蛋白结构决定了疫苗的有效性。
然而实验表面，天然的SARS-CoV-2的刺突蛋白S基因在人体内的表达量较低，如果以原始的S密码子来表达为抗原，不足以产生足够的抗原，其疫苗可能无效或是效价很低，不足以抵抗病毒的感染。
发明内容
本发明的目的在于克服现有技术的不足，提供一种优化的S蛋白核苷酸序列。该核苷酸序列在1)DNA质粒；2)整合到腺病毒载体之后，转染人细胞株后可以表达生产更多的S蛋白，有望作为核酸疫苗或重组病毒疫苗的抗原基因，用于预防SARS-CoV-2感染。
本发明所采取的技术方案是：
本发明的第一个方面，提供：
一种用于预防SARS-CoV-2感染的疫苗，所述疫苗包括：
a)如SEQ ID NO：1所示的核苷酸序列；或
b)与SEQ ID NO：1所示的核苷酸具有至少80％、85％、90％、95％、100％同源性的核苷酸序列。
在一些实例中，所述核苷酸序列可在人源细胞或人体内表达蛋白。
在一些实例中，所述蛋白可在人体内：
诱导免疫应答；或
产生生物报告分子；或
用于检测的追踪分子；或
调节基因功能；或
作为治疗性分子。
所述诱导免疫应答包括抗体与细胞介导的免疫应答；
在一些实例中，所述疫苗还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
在一些实例中，所述疫苗还包括至少一种对COVID-19有治疗作用的药物。
本发明的第二个方面，提供：
一种表达载体，所述的载体含有本发明第一方面所述的核苷酸序列。
在一些实例中，本发明第一方面所述的核苷酸序列转录方向与所述载体其它基因的转录方向相反。这样可以进一步保证表达得到的蛋白具有更高的纯度。
在一些实例中，所述的载体为DNA质粒或RNA表达质粒或病毒载体。
在一些实例中，所述的病毒载体为腺病毒载体。特别的，所述腺病毒载体为复制缺陷型Ad5载体。
本发明的第三个方面，提供：
本发明第一方面所述的疫苗的应用，所述应用包括：
制备COVID-19检测试剂；
制备基因功能调节剂。
本发明的第四个方面，提供：
一种预防或治疗COVID-19的方法，包括给予人预防量或治疗量的疫苗，所述疫苗如本发明第一方面所述。
本发明的有益效果是：
本发明一些实例的疫苗，其含有的S蛋白核苷酸序列易于在人体细胞表达，可以诱导产生更多的S蛋白，有望作为一种重组病毒疫苗，用于预防SARS-CoV-2感染。
本发明的一些实例，具有较好的安全性。
本发明一些实例的动物实验结果显示：免疫小鼠与猕猴后可产生高效价抗体及细胞免疫。接受疫苗单 次肌肉注射或喷鼻/滴口免疫的猕猴，在免疫后30天接受攻毒，可完全抵御病毒感染。接受单次1/10低剂量(1×10 10VP/只)疫苗肌肉注射的猕猴，在免疫后8周接受攻毒，也可完全抵御病毒感染。在疫苗组猕猴接受攻毒后，未观察到ADE或病毒引起的肺损伤。大多数接受疫苗的猕猴在攻毒后，抗体水平没有升高，提示病毒被迅速清除，对免疫系统没有产生刺激作用。与全病毒灭活苗不同，本发明除了刺激机体的体液免疫外，还产生细胞免疫，可能进一步提高对机体的保护作用。此外经鼻口免疫也能获得良好保护，提示粘膜免疫及非注射免疫的可行性。
附图说明
图1是含有密码子优化的S基因核苷酸序列的质粒转染人源细胞后表达S蛋白的检测结果。
图2是动物免疫原性评价的实验结果，表明Ad5-NB2能够诱异小鼠产生抗体。
图3是猕猴免疫后不同时间血清中结合抗体检测结果，a：猕猴免疫后不同时间血清中结合抗体检测结果b：猕猴免疫后18天血清中结合抗体检测结果。
图4是猕猴免疫后血清抗新型冠状病毒活病毒中和抗体滴度。
图5是猕猴免疫18天后外周血PBMCs细胞ELISpot检测结果。
图6是猕猴攻毒后咽拭子病毒基因组拷贝数测定，a：对照组攻毒后病毒基因组拷贝数变化b：肌注高剂量组攻毒后病毒基因组拷贝数变化c：肌注低剂量攻毒后病毒基因组拷贝数变化d：喷鼻+滴口攻毒后病毒基因组拷贝数变化。
图7是疫苗接种猕猴和非疫苗接种猕猴咽拭子中的峰值病毒载量，每个空心圆圈代表一只猕猴的峰值病毒载量。菱形为高剂量肌注接种的猕猴；方块为喷鼻+滴口接种的猕猴；三角形为低剂量肌注接种的猕猴；黑色圆圈为未接种疫苗的猕猴。
图8是猕猴攻毒后不同部位病毒基因组拷贝数测定。
图9是猕猴攻毒后肺部病理结果。a：对照组攻毒后猕猴肺部病理切片(HE染色)b：对照组未攻毒猕猴肺部病理切片(HE染色)c：肌注高剂量组(1×10 11VP)攻毒后肺部病理切片(HE染色)d：滴鼻+滴口联合免疫组(5×10 10vp/途径)攻毒后肺部病理切片(HE染色)e：肌注低剂量组(1×10 10VP)攻毒后肺部病理切片(HE染色)。
图10是猕猴攻毒前后抗SARS-CoV-2中和抗体变化。a：高剂量肌注组攻毒前后抗SARS-CoV-2中和抗体变化b：滴鼻/滴口组攻毒前后抗SARS-CoV-2中和抗体变化c：低剂量肌注组攻毒前后抗SARS-CoV-2中和抗体变化d：对照组攻毒前后抗SARS-CoV-2中和抗体变化。
图11是猕猴免疫前后抗Ad5中和抗体变化。a：高剂量肌注组免疫前后抗Ad5中和抗体变化。b：低剂量肌注组免疫前后抗Ad5中和抗体变化c：滴鼻/滴口组免疫前后抗Ad5中和抗体变化d：对照组免疫前后抗Ad5中和抗体变化。
具体实施方式
SARS-CoV-2的刺突蛋白(Spike protein，S)的氨基酸序列如YP_009724390.1所示，记为NB1。
真核细胞转录的mRNA前体能够通过不同剪接方式(选择不同的剪接位点组合)产生不同的mRNA剪接异构体的过程，最终导致同一个基因序列产生的不同的蛋白质。这对蛋白的表达是非常不利的。发明人通过对野生型的天然核苷酸序列进行密码子优化，同时基于自有技术去除潜在的可变剪切位点，保证了蛋白表达的唯一性，减少了蛋白后续纯化的难度。优化得到的核苷酸序列记为NB2，其具体序列如SEQ ID NO：1所示：
Figure PCTCN2020110085-appb-000001
Figure PCTCN2020110085-appb-000002
Figure PCTCN2020110085-appb-000003
为了降低基因的GC含量，发明人在NB2基础上进一步将密码子优化，合成的序列命名为NB4，具体如下SEQ ID NO.：2所示，NB4和NB2的序列相同性为85.93％。
Figure PCTCN2020110085-appb-000004
Figure PCTCN2020110085-appb-000005
Figure PCTCN2020110085-appb-000006
Spike基因表达载体的构建：
分别以NB1，NB2和NB4为模板，以NB1-F和NB1-R为引物PCR扩增获得NB1片段，以NB2-F和NB2-R为引物PCR扩增获得NB2片段，以NB4-F和NB4-R为引物PCR扩增获得NB4片段后，再分别以各CMV-R和BGH-F为引物，以本公司已有的pGA1-EGFP质粒为模板，PCR扩增载体质粒骨架pGA1后，采用同源重组酶(Exnase)分别与NB1，NB2和NB4片段进行体外二片段重组，得到pGA1-NB1，pGA1-NB2和pGA1-NB4。将pGA1-NB2采用Bstz17I和SgarAI线性化，采用BJ5183感受态与对应删除E1区唯一酶切位点酶切线性化的pAd5△E1△E3(即Ad5空载体)进行同源重组，构建pAd5-NB2疫苗载体。
扩增NB1的引物序列：
NB1-F：GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTTGTTTTTCTTGT(SEQ ID NO.：3)
NB1-R：AGAATAGGGCCCTCTAGACTAGTTTATGTGTAATGTAATTTG(SEQ ID NO.：4)
扩增NB2的引物序列：
NB2-F：GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTCGTGTTTCTGGT(SEQ ID NO.：5)
NB2-R：AGAATAGGGCCCTCTAGACTAGTTTATCAGGTGTAGTGCAGCTTC(SEQ ID NO.：6)
扩增NB4的引物序列：
NB4-F：GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTTGTCTTCCTGGT(SEQ ID NO.：7)
NB4-R：AGAATAGGGCCCTCTAGACTAGTTTAGGTGTAGTGCAGCTTC(SEQ ID NO.：8)
扩增pGA1的引物序列：
BGH-F:TCTAGAGGGCCCTATTCTATAGTGTC(SEQ ID NO.：9)
CMV-R:GGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACGCTAGAGTCCGG(SEQ ID NO.：10)
PCR条件：95℃，3min；95℃，30s；60℃ 30s；72℃ 2min；cycles 30；72℃，5min。
Ad5-NB2载体的拯救与生产
1)按照常规方法，pAd5-NB2以AsiSI线性化，乙醇沉淀回收，阳离子脂质体转染法转染293细胞，转染后8小时，加入2毫升含5％胎牛血清的DMEM培养基，孵育7-10天，观察细胞病变；
2)出毒后，收集细胞及培养上清，在37度水浴及液氮中反复冻融3次并离心去除细胞碎片，上清感染10厘米皿；
3)2-3天后，收集细胞及培养上清，反复冻融3次并离心去除细胞碎片，上清感染3-5个15厘米皿；
4)2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片；上清感染30个15厘米皿2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片；
5)上清加至氯化铯密度梯度离心管；4℃，40000转，离心4小时；
6)吸出病毒条带，脱盐，分装。
以OD260吸光度测定病毒粒子滴度，计算公式为：病毒浓度＝OD260×稀释倍数×36/基因组长度(Kb)；病毒储存液于-80℃冻存。
检测Spike基因表达：
按照常规方法，利用阳离子脂质体，分别将2.5ug的pGA-NB1，pGA-NB2和pGA-NB4转染48小时后，收集细胞。用Ad5-NB2和Ad5空载体病毒侵染HEK293细胞，24h后收集细胞。上述四个样品按照常规的Western Blot方法处理样品，并进行蛋白检测(图1)。
可以看出，pGA1-NB1样品中没有检测到S蛋白的表达，而经过密码子优化的pGA1-NB2、pGA1-NB4和疫苗候选株Ad5-NB2样品中能够观察到S蛋白的表达，说明NB2的序列具有意料之外的效果。pGA1-NB4也可以观察到S蛋白的表达，说明与NB2至少85％以上，进一步为86％、90％、95％、98％以上序列相同性的核苷酸序列均能有限表达相应的蛋白。
动物免疫原性评价
6-8周龄Balb/c小鼠分为5组，每组5只；第0天，分别肌注免疫Ad5-NB2剂量：5×109vp/只，采用肌注和滴鼻两种接种方式；第8天，眼眶取血并分离血清。采用酶联免疫吸附测定(ELISA)，以新冠病毒的S蛋白为抗原检测血清中的抗体水平。具体操作为：
1)96孔板，每孔加100ul的PBS和50ng S蛋白，4℃过夜，16-18小时；
2)吸掉上清，PBST洗一遍，加入200ul 5％脱脂牛奶室温封闭2h；
3)PBST洗2次；
4)加样：加入100ul稀释后的血清样品，37℃孵育2h，用PBST洗5次；
5)加酶标抗体：加入100ul稀释后HRP标记IgM或IgG二抗，37℃孵育2h；
6)PBST洗6-8次；
7)加底物液显色：加入100ul TMB显色；
8)终止反应：加入50ul 1M硫酸终止反应；
9)结果判定：测OD值，OD值控制在0.1-4。
实验结果表明，Ad5-NB2能够诱异小鼠产生抗体(图2)。
免疫保护性评估
猕猴来自广东蓝岛生物技术有限公司。接种疫苗的猕猴年龄为6～14岁。
随机分为4组，每组4只，具体如下表：
Figure PCTCN2020110085-appb-000007
备注：每只猕猴滴鼻和滴口的免疫剂量分别为5×10 10VP。
用Ad5-NB2毒株制备疫苗免疫猕猴，分别于免疫后12天、18天、24天取血采用ELISA法测定抗体结合效价，真病毒检测中和抗体滴度；分离外周血用ELISpot法检测细胞免疫反应。
实验结果：
(1)结合抗体
肌肉注射接种后12天，注射1×10 11VP的4只猕猴和注射1×10 10VP的3只猕猴血清中均能检测到显著的S特异性IgG。24天，所有免疫猕猴血清均可检测到S-特异性IgG。在接受低剂量或高剂量免疫接种的猕猴中，S特异性IgG随时间持续增加(图3a)。
滴鼻/滴口免疫接种12天后，在4只接种过疫苗的猕猴中，3只能检测到血清中S-特异性IgG。第24 天，所有免疫猕猴均可检测到血清中S-特异性IgG。
与肌肉注射相比，滴鼻/滴口免疫在接种后第18天，血清IgG滴度达到最高，比同等剂量的肌肉免疫猕猴低1～2个对数(图3a、b)。
(2)中和抗体
通过利用新型冠状病毒活病毒(2019-nCoV-WIV04毒株)与血清抗体的中和反应测定血清样品中的中和抗体滴度。
获得的动物血清样品在56℃热灭活30min，稀释至1:50、1:150、1:450、1:1350、1:4050和1:12150，加入等量的活病毒，在37℃，5％CO 2条件下培养3天后，用4％甲醛固定细胞然后用结晶紫染色。计数噬斑数量，确定中和抗体效价(EC 50)。
结果显示(图4)：免疫后28天，肌肉注射1×10 11VP的猕猴血清具有针对新型冠状病毒活病毒的中和抗体，滴鼻/滴口免疫(各5×10 10VP)的猕猴血清也具有针对新型冠状病毒活病毒的中和抗体，但低于肌肉注射免疫。免疫后56天，肌肉注射1×10 10VP的猕猴血清也具有针对新型冠状病毒活病毒的中和抗体，但低于1×10 10VP肌肉注射免疫的猕猴。
(3)细胞免疫
为了确定Ad5-NB2是否也能引起非人灵长类动物(NHPs)的细胞免疫反应，我们检测了外周血单个核细胞(PBMCs)中S-特异性IFN-γ分泌细胞对S1和S2肽库的反应。
结果表明：
1)肌注免疫接种后第18天，8只肌肉注射的猕猴全部对S1肽库有细胞免疫反应，6只对S2肽库有细胞免疫反应(图5)。
2)滴鼻/滴口免疫接种后第18天，4只接种的猕猴中，2只对S1肽库有弱的细胞免疫反应，第18天时对S2均无明显反应(图5)。
因此，在猕猴中，细胞免疫反应主要针对S1区，与在小鼠中观察到的现象相似。这一结果表明，疫苗肌注免疫可引起系统性细胞免疫反应对S蛋白的反应，特别是对S1区的反应，而在疫苗黏膜免疫接种引起的系统性细胞免疫反应要较弱。
保护性试验(猕猴攻毒试验)
攻毒试验在中国科学院武汉病毒研究所P4实验室进行。1×10 11VP免疫组(肌注组，滴鼻/滴口免疫组)在免疫后30天，1×10 10VP低剂量免疫组(肌注组)在免疫后8周，通过气管内接种2×10 4TCID50 SARS-CoV-2(2019-nCoV-WIV04)进行攻毒试验。因P4实验室空间限制，仅随机挑选3只高剂量组肌注猴，3只高剂量滴鼻+滴口免疫猴和3只低剂量肌注免疫猴进行攻毒试验。同时4只未接种疫苗的猕猴(C1-C4)也接受同样剂量病毒的攻毒试验，另外两只未免疫的D1，D2猕猴接受的攻毒剂量为400TCID50。攻毒后连续10天进行咽拭、支气管、肺叶等10个位点核酸检测。检测结果表明：
1)免疫组的猕猴攻毒后咽拭子新冠病毒基因组检测不到病毒或部分点检测到极低的拷贝数。
●没有接受疫苗免疫的猕猴(C1-C4，D1-D2)，咽拭子中能够检测到高载量新冠病毒核酸，攻毒后第5天，咽拭子病毒载量最高可达4.2×10 6拷贝/ml(图6a)。
●高剂量1×10 11VP肌注免疫后30天攻毒，咽拭子进行qPCR定量检测，结果显示，仅在第1天或第5天部分猕猴咽拭子略高于检测限，后续无法检出。因此，肌注免疫接种1×10 11VP的Ad5-NB2可以有效预防SARS-CoV-2的感染(图6b)。
●低剂量1×10 10VP肌注免疫后56天(8周)攻毒，咽拭子进行qPCR定量检测，结果显示，仅在第3天或第5天部分猕猴出现略高于检测限，后续无法检出。因此，肌注免疫接种1×10 10VP的Ad5-NB2能有效预防SARS-CoV-2的感染(图6c)。
●滴鼻/滴口联合免疫的猕猴在攻毒后前4天内均无法检出病毒，部分猕猴在第6天或第7天略有高于检出限检出，第10天降至无法检出。因此，滴鼻+滴口联合免疫也可有效抵御SARS-CoV-2的感染(图6d)。
综上所述，所有免疫的猕猴均获得完全保护，有效抵御SARS-CoV-2的感染。肌注免疫组疫苗在猕猴体内引起的全身抗体和细胞免疫反应显著高于黏膜免疫组疫苗。
2)所有接种过疫苗的猕猴在攻毒后均未发现抗体依赖性感染增强(ADE)的迹象。
我们根据曲线下面积(AUC)计算病毒载量。疫苗免疫的猕猴病毒AUC(2.7±0.6 log10)比未免疫猕猴的(6.1±1.0log10)低2500倍(见图7)。
3)对安乐死猕猴后9个解剖部位(包括气管、左支气管和右支气管、左肺和右肺的上、中、下部位)的活检样本中病毒基因组的存在情况进行评估。
结果显示，未接种的猕猴C3的左支气管(2.4×10 5拷贝/ml)和左肺下部(1.8×10 4拷贝/ml)检测到病毒基因组。在未接种疫苗的猕猴C4气管(4.7×10 4拷贝/ml)、左支气管(2.2×10 4拷贝/ml)和右支气管(1.8×10 4拷贝/ml)中也检测到病毒基因组。与此相反，从9只接种过疫苗的猕猴采集的81份活检样本新冠病毒核酸检测结果均为阴性(图8)。
4)病理切片证实免疫猕猴的肺部得到有效保护
对未接种疫苗和接种疫苗的猕猴的肺切片进行了组织病理学分析。在未接种SARS-CoV-2的猕猴中，气管内接种SARS-CoV-2可引起严重的间质性肺炎，表现为肺泡间隔扩张、多数肺泡内单核细胞和淋巴细胞浸润以及肺泡内一定比例的水肿。在接种过疫苗的猕猴中，攻毒实验未引起猕猴的明显的病理异常。部分猕猴肺部略有轻微的组织病理学变化，这可能是由于直接气管插管对猕猴进行攻毒造成的机械损伤(图9)。
5)免疫前后猕猴血清抗体效价变化
采用斑减少中和试验(PRNT)比较攻毒前后的血清中和抗体滴度。
●所有未接种疫苗的猕猴在攻毒前或攻毒后7天均未表现出血清中和SARS-CoV-2的活性(<1:50)(图10d)。
●高剂量肌注免疫组，攻毒前的血清中和抗体滴度分别为1:636、1:389、1:784；攻毒后第7天，其中和抗体滴度分别为1:1225、1:518和1:1350，表明攻毒没有或增强作用较弱(图10a)。
●低剂量肌注免疫组，与攻毒前相比，攻毒第7天，只有1只猕猴(编号080066)的血清中和抗体滴度显著升高(IC50 1:188→1:1460)，另外2只猕猴血清中和抗体滴度并无升高(IC50 1:315→1:192，1:400→1:330)(图10c)。
●滴鼻/滴口免疫组，攻毒前与攻毒后第7天的血清中和抗体滴度都比较低，没有显著变化。攻毒前的血清中和抗体滴度分别为1:164、1:137、1:150；攻毒后第7天，其中和抗体滴度分别为1:193、1:168和1:130(图10b)。
●对Ad5-NB2免疫猕猴的体内抗Ad5中和抗体水平进行检测。结果表明肌肉注射组免疫Ad5-NB2后，血清中的Ad5中和抗体滴度在第6天时已升高。然而滴鼻/滴口免疫组接受Ad5-NB2后，血清中的Ad5中和抗体滴度在第6天时尚无显著升高，至第24天时也升高但并不高于低剂量免疫组(图11a～d)。
综上所述，攻毒前后免疫组的9只猕猴中有6只的抗新冠病毒的中和抗体滴度无明显升高，提示病毒被立即消除，没有出现明显的病毒复制的可能性。
小结：该复制缺陷型5型腺病毒载体优化设计的S基因NB2，可显著提高抗原表达，免疫小鼠与猕猴后可产生高效价抗体及细胞免疫。接受疫苗单次肌肉注射或喷鼻/滴口免疫的猕猴，在免疫后30天接受攻毒，可完全抵御病毒感染。接受单次1/10低剂量(1×10 10VP/只)疫苗肌肉注射的猕猴，在免疫后8周接受攻毒，也可完全抵御病毒感染。在疫苗组猕猴接受攻毒后，未观察到ADE或病毒引起的肺损伤。大多数接受疫苗的猕猴在攻毒后，抗体水平没有升高，提示病毒被迅速清除，对免疫系统没有产生刺激作用。与全病毒灭活苗不同，该疫苗除了刺激机体的体液免疫外，还产生细胞免疫，可能进一步提高对机体的保护作用。此外经鼻口免疫也能获得良好保护，提示粘膜免疫及非注射免疫的可行性。

Claims (11)

  1. 一种用于预防SARS-CoV-2感染的疫苗，其特征在于：所述疫苗包括：
    如SEQ ID NO：1所示的核苷酸序列；或
    与SEQ ID NO：1所示的核苷酸具有至少80％、85％、90％、95％、100％同源性的核苷酸序列。
  2. 根据权利要求1所述的疫苗，其特征在于：所述核苷酸序列可在人源细胞或人体内表达蛋白。
  3. 根据权利要求2所述疫苗，其特征在于：所述蛋白可在人体内：
    诱导免疫应答；或
    产生生物报告分子；或
    用于检测的追踪分子；或
    调节基因功能；或
    作为治疗性分子。
  4. 根据权利要求1～3任一项所述疫苗，其特征在于：还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
  5. 根据权利要求1～3任一项所述疫苗，其特征在于：还包括至少一种对COVID-19有治疗作用的药物。
  6. 一种表达载体，其特征在于：所述的载体含有权利要求1所述的核苷酸序列。
  7. 权利要求6所述的表达载体，其特征在于：所述的载体为DNA质粒、RNA表达质粒或病毒载体。
  8. 权利要求7所述的表达载体，其特征在于：所述的病毒载体为腺病毒载体。
  9. 根据权利要求8所述的腺病毒载体，其特征在于：权利要求1所述的核酸分子转录方向与所述载体其它基因的转录方向相反。
  10. 权利要求1所述的疫苗的应用，所述应用包括：
    制备COVID-19检测试剂；
    制备基因功能调节剂。
  11. 一种预防或治疗COVID-19的方法，包括给予人预防量或治疗量的疫苗，所述疫苗如权利要求1～5任一项所述。
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