WO2021184988A1 - 一种用于预防SARS-CoV-2感染的Ad35载体疫苗 - Google Patents

一种用于预防SARS-CoV-2感染的Ad35载体疫苗 Download PDF

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WO2021184988A1
WO2021184988A1 PCT/CN2021/074838 CN2021074838W WO2021184988A1 WO 2021184988 A1 WO2021184988 A1 WO 2021184988A1 CN 2021074838 W CN2021074838 W CN 2021074838W WO 2021184988 A1 WO2021184988 A1 WO 2021184988A1
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vector
vaccine
nucleic acid
acid sequence
seq
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陈凌
关素华
杨臣臣
汪乾
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广州恩宝生物医药科技有限公司
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Priority to US17/598,624 priority Critical patent/US20220331420A1/en
Priority to EP21751950.3A priority patent/EP3912638A4/en
Publication of WO2021184988A1 publication Critical patent/WO2021184988A1/zh

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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
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    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to an Ad35 vector vaccine for preventing SARS-CoV-2 infection.
  • the new crown pneumonia epidemic is very severe and has spread in many countries around the world. As of March 6, 2020, there have been more than 100,000 confirmed cases of new coronary pneumonia worldwide. However, for the new coronavirus, there are currently no specific antiviral drugs and preventive vaccines that have been clearly verified at home and abroad. Therefore, preventing the spread of the virus is the key to controlling the epidemic. Vaccines are the most cost-effective and effective intervention to prevent and control new coronavirus infections.
  • the published results of more than one hundred SARS-CoV-2 virus genome comparisons show that the overall degree of mutation of the virus is low, and no recombination phenomenon has occurred. Therefore, if the SARS-CoV-2 vaccine is successfully developed, it will be able to protect the population from the new coronavirus infection, thereby suppressing the outbreak of new epidemics.
  • S-protein that forms the "crown" is an obvious target and has become the focus of most research teams.
  • the full sequence of SARS-CoV-2 is shown in NC_045512.2.
  • the 21563..25384 nucleic acid of this sequence is the coding sequence of Spike protein (S), and the full length of S protein is 1273aa.
  • S protein plays an important role in mediating the binding of virus particles to host cell receptors and inducing neutralizing antibodies.
  • vaccines with S protein as the antigen including nucleic acid vaccines, subunit vaccines, and viral vector vaccines are very promising to prevent SARS-CoV-2 infection, but the expression level and protein structure of the S protein in these vaccines determine the vaccine’s effectiveness. Effectiveness.
  • Adenovirus is a commonly used vector in vaccine development and gene therapy, and is widely used in the field of biomedicine. Compared with other viral vectors, adenovirus is less toxic, and infection with adenovirus only causes mild cold symptoms. At present, most of the adenovirus vector vaccines developed by research teams are based on type 5 adenovirus. The proportion of pre-existing antibodies against type 5 adenovirus vectors in the population is very high, which affects the type 5 adenovirus vector vaccine titer to a certain extent.
  • the purpose of the present invention is to overcome at least one shortcoming of the prior art and provide an Ad35 vector vaccine that is expected to be used to prevent SARS-CoV-2 infection.
  • the invention integrates the optimized S gene into the replication-deficient Ad35 vector, infects cells with adenovirus, presents the neocoronavirus antigen, and makes the body produce a specific immune response after immunization, thereby blocking the neocoronavirus infection.
  • Ad35 has a lower proportion of pre-existing antibodies in the population than Ad5, so it can prevent the carrier from being neutralized by the pre-existing antibodies in the human body, and the immune effect is expected to be higher than that of the Ad5 carrier vaccine.
  • the replication ability of Ad35 is higher than that of Ad5.
  • the cost of Ad35 vector vaccine is expected to be lower than that of Ad5 vector vaccine.
  • Ad35 vector vaccine for preventing SARS-CoV-2 infection, comprising an Ad35 vector carrying a nucleic acid sequence shown in SEQ ID NO:1.
  • the Ad35 vector is a replication-defective Ad35 vector.
  • the replication-defective Ad35 vector is a replication-defective Ad35 vector lacking genes in the E1 and E3 regions.
  • the replication of the Ad35 vector in the cell can be effectively avoided, and the safety of the product can be improved.
  • the transcription direction of the nucleic acid sequence shown in SEQ ID NO:1 is opposite to the transcription direction of other genes in the Ad35 vector. In this way, the nucleic acid sequence shown in SEQ ID NO: 1 can be better expressed, which is beneficial to improve the safety and potency of the product.
  • the vector can express the nucleic acid sequence shown in SEQ ID NO:1 more specifically, increasing the potency of the vaccine and improving its safety.
  • the Ad35 vector has elements that regulate the expression of the nucleic acid sequence shown in SEQ ID NO:1. In this way, the expression of the nucleic acid sequence shown in SEQ ID NO:1 can be more artificially regulated to obtain better safety.
  • the nucleic acid sequence can express the protein in a cell of human origin or in a human body.
  • the protein can be in the human body:
  • the Ad35 carrier vaccine further includes at least one of a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.
  • Adjuvants, carriers, diluents or excipients can be selected according to the specific dosage form of the vaccine.
  • the dosage form of the Ad35 carrier vaccine includes, but is not limited to, injection, oral, aerosol inhalation and other common vaccine dosage forms.
  • the adenovirus vector vaccine can also be used in combination with other vaccines.
  • the Ad35 vector vaccine also includes at least one drug that has a therapeutic effect on COVID-19.
  • Some examples of the present invention have better safety and are convenient to use. Experiments show that it can produce more S protein in human cells. It is expected to be developed as a vaccine for preventing SARS-CoV-2 infection.
  • Some examples of the present invention can be used in combination with other vaccines, and can also be used as therapeutic vaccines for new coronary pneumonia. They can be vaccinated at the initial stage of infection to rapidly induce immune responses in the human body and achieve therapeutic effects.
  • Figure 1 is the detection result of S protein expression.
  • Figure 2 shows the detection results of binding antibodies in the serum of rhesus monkeys at different times after immunization.
  • Figure 3 shows the results of ELISpot detection of PBMCs in peripheral blood of rhesus monkeys 18 days after immunization.
  • the amino acid sequence of the spike protein (S) of SARS-CoV-2 is shown in YP_009724390.1, with a full length of 1273aa, denoted as NB1.
  • the mRNA precursors transcribed by eukaryotic cells can produce different mRNA splicing isoforms through different splicing methods (selecting different splicing site combinations), which ultimately leads to different proteins produced by the same gene sequence. This is very detrimental to protein expression.
  • the inventors By optimizing the codon of the wild-type natural nucleic acid sequence and removing potential variable splicing sites based on its own technology, the inventors ensure the uniqueness of protein expression and reduce the difficulty of subsequent purification of the protein.
  • the optimized nucleic acid sequence is denoted as NB2, and its specific sequence is shown in SEQ ID NO:1:
  • Ad35 expression S protein vector pAd35-NB2 Construction of Ad35 expression S protein vector pAd35-NB2:
  • NB1 and NB2 were used as templates, NB1-F and NB1-R were used as primers for PCR amplification to obtain NB1 fragments, and NB2-F and NB2-R were used as primers for PCR amplification to obtain NB2 fragments.
  • BGH-F as primers, using pGA351-EGFP plasmid as template, after PCR amplification of vector plasmid backbone pGA351, homologous recombinase (Exnase) was used to recombine with NB1 and NB2 fragments in vitro to obtain pGA351-NB1 and pGA351 -NB2.
  • the pGA351-NB2 was linearized with Bstz17I and SgarAI, and the linearized pAd35 ⁇ E1 ⁇ E3 (that is, the Ad35 empty vector) was digested with BJ5183 competent and corresponding to delete the only restriction site in the E1 region for homologous recombination to construct pAd35-NB2 Vaccine carrier.
  • the primer sequence for amplifying NB1 is the primer sequence for amplifying NB1:
  • NB1-F GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTTGTTTTTCTTGT (SEQ ID NO.: 2)
  • NB1-R AGAATAGGGCCCTCTAGACTAGTTTATGTGTAATGTAATTTG (SEQ ID NO.: 3)
  • the primer sequence for amplifying NB2 is the primer sequence for amplifying NB2
  • NB2-F GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTCGTGTTTCTGGT (SEQ ID NO.: 4)
  • NB2-R AGAATAGGGCCCTCTAGACTAGTTTATCAGGTGTAGTGCAGCTTC (SEQ ID NO.: 5)
  • the primer sequence for amplifying pGA351 is the primer sequence for amplifying pGA351
  • BGH-F TCTAGAGGGCCCTATTCTATAGTGTC (SEQ ID NO.: 6)
  • CMV-R GGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACGCTAGAGTCCGG (SEQ ID NO.: 7)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C 2min; cycles 30; 72°C, 5min.
  • pAd35-NB2 was linearized with AsiSI, recovered by ethanol precipitation, and transfected into 293 cells by cationic liposome transfection;
  • virus concentration OD260 ⁇ dilution factor ⁇ 36/genome length (Kb); virus stock solution is frozen at -80°C.
  • the macaques are from Guangdong Landao Biotechnology Co., Ltd.
  • the age of the vaccinated macaques is 2-3 years old. Randomly divide into 3 groups, the experimental group has 2 animals in each group, and the control group has 4 animals. The details are as follows:
  • the Ad35-NB2 strain was used to prepare vaccines to immunize rhesus monkeys. Blood was collected 14 days and 18 days after immunization, and the antibody binding titer was determined by EL ISA method. Peripheral blood was separated and the cellular immune response was detected by ELISpot method.
  • the antibody titer induced by intramuscular immunization and nasal drip immunization is not much different.
  • Ad35-NB2 can also cause cellular immune responses in non-human primates (NHPs).
  • NEPs non-human primates
  • PBMCs peripheral blood mononuclear cells
  • the cellular immune response is mainly directed at the S1 region. This result indicates that intramuscular injection of vaccine can cause systemic cellular immune response to S protein, especially to the S1 area, while the systemic cellular immune response caused by vaccine mucosal immunization is weaker.
  • the above experimental results show that the vaccine can stimulate the rhesus monkeys to produce cellular immunity, which may further enhance the protection of the body.

Abstract

提供了一种用于预防SARS-CoV-2感染的Ad35载体疫苗，该疫苗包括负载有SEQ ID NO：1所示核酸序列的Ad35载体。该疫苗可以和其他疫苗联用，也可作为新冠肺炎的治疗性疫苗。

Description

一种用于预防SARS-CoV-2感染的Ad35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Ad35载体疫苗。
背景技术
新冠肺炎疫情非常严峻，已在全球多国蔓延。截至2020年3月6日，全球新冠肺炎确诊病例超过10万例。然而，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。疫苗是预防和控制新型冠状病毒感染最经济有效的干预措施。已公布的一百多个SARS-CoV-2病毒基因组比对的结果显示病毒整体突变程度较低，未发生重组现象。因此，如果SARS-CoV-2疫苗研发成功，必将能够保护人群免受新冠病毒的感染，从而抑制新疫情的暴发。
SARS-CoV-2冠状病毒的病毒颗粒结构中，组成“皇冠”的S-蛋白是一个明显的靶点，成为大多数研究团队研究的重点。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蛋白全长1273aa。已有研究团队通过对S蛋白三维结构计算机模拟，成功地揭示了S蛋白与其侵入细胞过程中的受体ACE2的关系。S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是非常有希望预防SARS-CoV-2感染的，但是这些疫苗中S蛋白的表达水平、蛋白结构决定了疫苗的有效性。
然而实验表明，天然的SARS-CoV-2的刺突蛋白S基因在人肾细胞HEK293表达水平很低，因此如果以原始的S密码子来表达为抗原，其疫苗可能无效或是效价很低，不足以抵抗病毒的感染。
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。与其它病毒载体比，腺病毒低毒性，感染腺病毒只引起轻微的感冒症状。目前大多数研究团队开发的腺病毒载体疫苗都是基于5型腺病毒。人群中针对5型腺病毒载体的预存抗体比例很高，一定程度影响5型腺病毒载体疫苗效价。
发明内容
本发明的目的在于克服现有技术的至少一个不足，提供一种有望用于预防 SARS-CoV-2感染的Ad35载体疫苗。
本发明通过将优化的S基因整合到复制缺陷型Ad35载体上，通过腺病毒感染细胞，呈递新冠病毒抗原，免疫后使机体产生特异性免疫反应，从而阻断新冠病毒的感染。Ad35在人群中预存抗体的比例低于Ad5，因此可避免载体被人体内预存抗体中和，免疫效果有望高于Ad5载体疫苗。同时Ad35复制能力高于Ad5，在疫苗生产方面，Ad35载体疫苗的成本有望低于Ad5载体疫苗。
本发明所采取的技术方案是：
一种用于预防SARS-CoV-2感染的Ad35载体疫苗，包括Ad35载体，所述Ad35载体中负载有SEQ ID NO：1所示的核酸序列。
在一些实例中，所述Ad35载体为复制缺陷型Ad35载体。
在一些实例中，所述复制缺陷型Ad35载体为缺失E1和E3区基因的复制缺陷型Ad35载体。
通过使用复制复制缺陷型Ad35载体，可以有效避免Ad35载体在细胞内的复制，提高产品的安全性。
在一些实例中，SEQ ID NO：1所示核酸序列的转录方向与所述Ad35载体其它基因的转录方向相反。这样可以更好地表达SEQ ID NO：1所示核酸序列，有利于提高产品的安全性的效价。
这样可以使载体更为专一地表达SEQ ID NO：1所示核酸序列，提高疫苗的效价同时提高其安全性。
在一些实例中，所述Ad35载体具有调控SEQ ID NO：1所示核酸序列表达的元件。这样可以更为人为调控SEQ ID NO：1所示核酸序列表达，以获得更佳的安全性。
在一些实例中，所述核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。
在一些实例中，所述的Ad35载体疫苗还包括药学上可接受的佐剂、载体、稀 释剂或赋形剂中的至少一种。佐剂、载体、稀释剂或赋形剂可以根据疫苗的具体剂型进行相应的选择。
在一些实例中，所述的Ad35载体疫苗的剂型包括但不限于注射剂、口服剂、气雾吸入剂等常见的疫苗剂型。
在一些实例中，所述腺病毒载体疫苗还可以和其他疫苗联用。
在一些实例中，所述的Ad35载体疫苗还包括至少一种对COVID-19有治疗作用的药物。
本发明的有益效果是：
本发明的一些实例，具有较好的安全性，使用方便。实验表明其在人体细胞中可以产生更多的S蛋白。有望开发为用于预防SARS-CoV-2感染的疫苗。
本发明的一些实例，可以和其他疫苗联用，也可作为新冠肺炎的治疗性疫苗，在患者被感染初期接种，快速诱导人体内免疫反应，达到治疗效果。
附图说明
图1是S蛋白表达的检测结果。
图2是猕猴免疫后不同时间血清中结合抗体检测结果。
图3是猕猴免疫18天后外周血PBMCs细胞ELISpot检测结果。
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，全长1273aa，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。优化得到的核酸序列记为NB2，其具体序列如SEQ ID NO：1所示：
Figure PCTCN2021074838-appb-000001
Figure PCTCN2021074838-appb-000002
Figure PCTCN2021074838-appb-000003
Ad35表达S蛋白载体pAd35-NB2的构建：
分别以NB1和NB2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片段后，再分别以各CMV-R和BGH-F为引物，以pGA351-EGFP质粒为模板，PCR扩增载体质粒骨架pGA351后，采用同源重组酶(Exnase)分别与NB1和NB2片段进行体外二片段重组，得到pGA351-NB1和pGA351-NB2。将pGA351-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pAd35△E1△E3(即Ad35空载体)进行同源重组，构建pAd35-NB2疫苗载体。
扩增NB1的引物序列：
NB1-F：GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTTGTTTTTCTTGT(SEQ ID NO.：2)
NB1-R：AGAATAGGGCCCTCTAGACTAGTTTATGTGTAATGTAATTTG(SEQ ID NO.：3)
扩增NB2的引物序列：
NB2-F：GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCACCATGTTCGTGTTTCTGGT(SEQ ID NO.：4)
NB2-R：AGAATAGGGCCCTCTAGACTAGTTTATCAGGTGTAGTGCAGCTTC(SEQ ID NO.：5)
扩增pGA351的引物序列：
BGH-F：TCTAGAGGGCCCTATTCTATAGTGTC(SEQ ID NO.：6)
CMV-R：GGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACGCTAGAGTCCGG(SEQ ID NO.：7)
PCR条件：95℃，3min；95℃，30s；60℃ 30s；72℃ 2min；cycles 30；72℃，5min。
Ad35-NB2载体的拯救与生产
1)按照常规方法，pAd35-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；
2)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；
3)出毒后，收集细胞及培养上清，在37℃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；
4)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；
5)2～3天后，收集细胞，反复冻融3次并离心去除细胞碎片；
6)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；
7)上清加至氯化铯密度梯度离心管；4℃，40000转，离心4小时；吸出病毒条带，脱盐，分装；
8)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2.5μg的pGA351-NB1和pGA351-NB2，转染A549细胞48小时后，收集细胞。用Ad35-NB2病毒侵染A549细胞，36h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)。
从图1可以看出，pGA351-NB1样品中没有检测到S蛋白的表达，而经过密码子优化的pGA351-NB2和疫苗候选株Ad35-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。
免疫原性评估：
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2-3岁。随机分为3组，实验组每组2只，对照组4只，具体如下表：
Figure PCTCN2021074838-appb-000004
用Ad35-NB2毒株制备疫苗免疫猕猴，分别于免疫后14天、18天取血采用EL ISA法测定抗体结合效价，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14、18天，肌肉注射1×10 11VP的所有猕猴血清中均能检测到显著的S和RBD特异性IgG，免疫组2只中，有1只可检测出显著的针对S2的IgG(图2)。
滴鼻免疫接种14天后，接种1×10 11VP的所有猕猴血清中均能检测到显著的S和RBD特异性IgG，针对S2的IgG相对较弱，但也能被检测(图2)。
肌注免疫和滴鼻免疫所诱导产生的抗体效价相差不大。
(2)细胞免疫
为了确定Ad35-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，肌肉注射1×10 11VP的所有猕猴全部对S1和S2肽库有细胞免疫反应(图3)。
2)滴鼻免疫接种后第18天，2只接种的猕猴中，1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3)。
因此，在猕猴中，细胞免疫反应主要针对S1区。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。
以上实验结果表明，疫苗可刺激猕猴产生细胞免疫，可能进一步提升对机体的保护作用。

Claims (9)

  1. 一种用于预防SARS‐CoV‐2感染的Ad35载体疫苗，包括Ad35载体，所述Ad35载体中负载有SEQ ID NO：1所示的核酸序列。
  2. 根据权利要求1所述的Ad35载体疫苗，其特征在于：所述Ad35载体为复制缺陷型Ad35载体。
  3. 根据权利要求2所述的Ad35载体疫苗，其特征在于：所述复制缺陷型Ad35载体为缺失E1和E3区基因的复制缺陷型Ad35载体。
  4. 根据权利要求1～3任一项所述的Ad35载体疫苗，其特征在于：所述Ad35载体具有调控SEQ ID NO：1所示核酸序列表达的元件。
  5. 根据权利要求1～3任一项所述的Ad35载体疫苗，其特征在于：SEQ ID NO：1所示核酸序列的转录方向与所述Ad35载体其它基因的转录方向相反。
  6. 根据权利要求1～3任一项所述的Ad35载体疫苗，其特征在于：所述核酸序列可在人源细胞或人体内表达蛋白。
  7. 根据权利要求6所述的Ad35载体疫苗，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。
  8. 根据权利要求1～3任一项所述的Ad35载体疫苗，其特征在于：还包括药学上可接受的佐剂、载体、稀释剂或赋形剂中的至少一种。
  9. 根据权利要求1～3任一项所述的Ad35载体疫苗，其特征在于：还包括至少一种对COVID-19有治疗作用的药物。
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