中文版 | English
题名

新型冠状病毒DNA疫苗的免疫原性和保护效果研究

其他题名
Study on the immunogenicity and protective effect of a novel coronavirus DNA vaccine
姓名
姓名拼音
HOU Mengqi
学号
11930141
学位类型
硕士
学位专业
0710 生物学
学科门类/专业学位类别
07 理学
导师
王鹏
导师单位
药理学系
论文答辩日期
2022-04-26
论文提交日期
2022-06-23
学位授予单位
南方科技大学
学位授予地点
深圳
摘要
新型冠状病毒(SARS-COV-2)是已知能感染人类的第七种冠状病毒, 人类感染后导致的新型冠状病毒肺炎(COVID-19)给全球公共卫生带来了 极大的挑战。作为一种正链 RNA 病毒,其变异速率快,从最早发现的野生 毒株到近期大流行的奥密克戎毒株,其传染能力逐渐增加,这使得疫情的 防控难度加大。研究发现新冠疫苗的接种显著降低了疾病的严重程度,因此急需研发有效的新冠病毒疫苗。核酸疫苗被认为是最有效的疫苗,DNA 疫苗是核酸疫苗的一种,相比其他疫苗,其具有众多优势:安全,廉价, 有效,稳定。
研究发现,S 蛋白上的 6 个突变会使 S 蛋白更加稳定,鉴于 DNA 疫苗 的优势和 S-6P 突变体蛋白的特点,本项目拟开发一种基于 S-6P DNA 疫 苗 , 以 期 用 于 新 冠 病 毒 的 防 控 。 在 疫 苗 设 计 上 , 我 们 将 F817A892A899A942K986 V987 六个氨基酸突变为脯氨酸。同时,我们还引 入了 D614G 这一关键位点的突变,因为 D614G 突变导致病毒的感染能力大大增强。S 全长蛋白包含 1273 个氨基酸,其中 C 末端(1214-1273)为 跨膜结构域,因此仅选择了 S 蛋白表面可及、最具抗原性的胞外结构域 (S1+S21-1213)为疫苗抗原。
在实验中,我们首先将 S1+S2 抗原基因重组克隆到已被美国 FDA 批准 用于核酸疫苗的 pVAX1 载体上,然后转化 DH5α感受态细胞进行质粒扩增, 并转染 293T 细胞验证课题设计的 DNA 疫苗能按预期翻译目标抗原,最后 疫苗质粒通过电穿孔方式免疫 Balb/c 小鼠,取小鼠免疫后血清经 ELISA 实 验评价疫苗所诱导的抗体,经竞争性中和抗体检测试剂盒和假病毒中和实 验检测抗体的病毒中和活性。实验结果显示我们成功构建了项目所设计的 DNA 疫苗,该疫苗免疫小鼠可以诱导产生高效价的 RBD 特异性抗体,及 对野生型假病毒具有较高活性的中和抗体。该课题的研究为今后 DNA 疫苗 的研发和探索提供一定的思路和方法。
关键词
语种
中文
培养类别
独立培养
入学年份
2019
学位授予年份
2022-07
参考文献列表

[1] Rabaan AA, Al-Ahmed SH, Haque S, et al. SARS-CoV-2, SARS-CoV, and MERS- COV: A comparative overview. Infez Med. 2020;28(2):174-184.
[2] Kirtipal N, Bharadwaj S, Kang SG. From SARS to SARS-CoV-2, insights on structure, pathogenicity and immunity aspects of pandemic human coronaviruses. Infect Genet Evol. 2020;85:104502. doi:10.1016/j.meegid.2020.104502
[3] Wrobel AG, Benton DJ, Xu P, et al. SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects [published correction appears in Nat Struct Mol Biol. 2020 Oct;27(10):1001]. Nat Struct Mol Biol. 2020;27(8):763-767. doi:10.1038/s41594-020-0468-7
[4] Zhang T, Wu Q, Zhang Z. Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak [published correction appears in Curr Biol. 2020 Apr 20;30(8):1578]. Curr Biol. 2020;30(7):1346-1351.e2. doi:10.1016/j.cub.2020.03.022
[5] Kumar S, Thambiraja TS, Karuppanan K, Subramaniam G. Omicron and Delta variant of SARS-CoV-2: A comparative computational study of spike protein. J Med Virol. 2022;94(4):1641-1649. doi:10.1002/jmv.27526
[6] Khailany RA, Safdar M, Ozaslan M. Genomic characterization of a novel SARS- CoV-2. Gene Rep. 2020;19:100682. doi:10.1016/j.genrep.2020.100682
[7] Naqvi AAT, Fatima K, Mohammad T, et al. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach. Biochim Biophys Acta Mol Basis Dis. 2020;1866(10):165878. doi:10.1016/j.bbadis.2020.165878
[8] Kadam SB, Sukhramani GS, Bishnoi P, Pable AA, Barvkar VT. SARS- CoV-2, the pandemic coronavirus: Molecular and structural insights. J Basic Microbiol. 2021;61(3):180-202. doi:10.1002/jobm.202000537
[9] 郁文亮,李家璜,华子春.新型冠状病毒的生物学特征及其药物研发策略[J]. 药 物 生 物 技 术 ,2020,27(01):1-7.DOI:10.19526/j.cnki.1005- 8915.20200101.
[10] 谢艳颖,胡雪峰.2019 新型冠状病毒(SARS-CoV-2)的结构和致病机制概 述[J].生物学教学,2021,46(04):4-7.
[11] Rahimi A, Mirzazadeh A, Tavakolpour S. Genetics and genomics of SARS-CoV-2: A review of the literature with the special focus on genetic diversity and SARS-CoV-2 genome detection. Genomics. 2021;113(1Pt 2):1221-1232. doi:10.1016/j.ygeno.2020.09.059
[12] Pan P, Shen M, Yu Z, et al. SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation [published correction appears in Nat Commun. 2021 Aug 31;12(1):5306]. Nat Commun. 2021;12(1):4664. Published 2021 Aug 2. doi:10.1038/s41467-021-25015-6
[13] Bhat EA, Khan J, Sajjad N, et al. SARS-CoV-2: Insight in genome structure, pathogenesis and viral receptor binding analysis - An updated review. Int Immunopharmacol. 2021;95:107493. doi:10.1016/j.intimp.2021.107493
[14] Lu S, Ye Q, Singh D, et al. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein. Nat Commun. 2021;12(1):502. Published 2021 Jan 21. doi:10.1038/s41467- 020-20768-y
[15] Cao Y, Yang R, Lee I, et al. Characterization of the SARS-CoV-2 E Protein: Sequence, Structure, Viroporin, and Inhibitors [published correction appears in Protein Sci. 2021 Dec;30(12):2482]. Protein Sci. 2021;30(6):1114-1130. doi:10.1002/pro.4075
[16] Bai Z, Cao Y, Liu W, Li J. The SARS-CoV-2 Nucleocapsid Protein and Its Role in Viral Structure, Biological Functions, and a Potential Target for Drug or Vaccine Mitigation. Viruses. 2021;13(6):1115. Published 2021 Jun 10. doi:10.3390/v13061115
[17] Hatmal MM, Alshaer W, Al-Hatamleh MAI, et al. Comprehensive Structural and Molecular Comparison of Spike Proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, and Their Interactions with ACE2. Cells. 2020;9(12):2638. Published 2020 Dec 8. doi:10.3390/cells9122638
[18] Sternberg A, Naujokat C. Structural features of coronavirus SARS-CoV-2 spike protein: Targets for vaccination. Life Sci. 2020;257:118056. doi:10.1016/j.lfs.2020.118056
[19] Xia X. Domains and Functions of Spike Protein in Sars-Cov-2 in the Context of Vaccine Design. Viruses. 2021;13(1):109. Published 2021 Jan 14. doi:10.3390/v13010109
[20] Johnson BA, Xie X, Bailey AL, et al. Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis. Nature. 2021;591(7849):293-299. doi:10.1038/s41586-021-03237-4
[21] Johnson BA, Xie X, Kalveram B, et al. Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis. Preprint. bioRxiv. 2020;2020.08.26.268854. Published 2020 Aug 26. doi:10.1101/2020.08.26.268854
[22] Segreto R, Deigin Y. The genetic structure of SARS-CoV-2 does not rule out a laboratory origin: SARS-COV-2 chimeric structure and furin cleavage site might be the result of genetic manipulation. Bioessays. 2021;43(3):e2000240. doi:10.1002/bies.202000240
[23] Cai Y, Zhang J, Xiao T, et al. Distinct conformational states of SARS-CoV-2 spike protein. Science. 2020;369(6511):1586-1592. doi:10.1126/science.abd4251
[24] Lan J, Ge J, Yu J, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020;581(7807):215-220. doi:10.1038/s41586-020-2180-5
[25] Thunders M, Delahunt B. Gene of the month: TMPRSS2 (transmembrane serine protease 2). J Clin Pathol. 2020;73(12):773-776. doi:10.1136/jclinpath-2020-206987
[26] Scialo F, Daniele A, Amato F, et al. ACE2: The Major Cell Entry Receptor for SARS-CoV- 2. Lung. 2020;198(6):867-877. doi:10.1007/s00408-020-00408-4
[27] Trougakos IP, Stamatelopoulos K, Terpos E, et al. Insights to SARS-CoV-2 life cycle, pathophysiology, and rationalized treatments that target COVID-19 clinical complications. J Biomed Sci. 2021;28(1):9. Published 2021 Jan 12. doi:10.1186/s12929-020-00703-5
[28] Baughn LB, Sharma N, Elhaik E, Sekulic A, Bryce AH, Fonseca R. Targeting TMPRSS2 in SARS-CoV-2 Infection. Mayo Clin Proc. 2020;95(9):1989-1999. doi:10.1016/j.mayocp.2020.06.018
[29] Ashraf UM, Abokor AA, Edwards JM, et al. SARS-CoV-2, ACE2 expression, and systemic organ invasion. Physiol Genomics. 2021;53(2):51-60. doi:10.1152/physiolgenomics.00087.2020
[30] Yesudhas D, Srivastava A, Gromiha MM. COVID-19 outbreak: history, mechanism, transmission, structural studies and therapeutics. Infection. 2021;49(2):199-213. doi:10.1007/s15010-020- 01516-2
[31] Plante JA, Liu Y, Liu J, et al. Spike mutation D614G alters SARS-CoV-2 fitness [published correction appears in Nature. 2021 Jul;595(7865):E1]. Nature. 2021;592(7852):116-121. doi:10.1038/s41586-020-2895-3
[32] Wang P, Nair MS, Liu L, et al. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature. 2021;593(7857):130-135. doi:10.1038/s41586-021-03398-2
[33] Kandeel M, Mohamed MEM, Abd El-Lateef HM, Venugopala KN, El-Beltagi HS. Omicron variant genome evolution and phylogenetics. J Med Virol. 2022;94(4):1627-1632. doi:10.1002/jmv.27515
[34] Araf Y, Akter F, Tang YD, et al. Omicron variant of SARS-CoV-2: Genomics, transmissibility, and responses to current COVID-19 vaccines [published online ahead of print, 2022 Jan 12]. J Med Virol. 2022;10.1002/jmv.27588. doi:10.1002/jmv.27588
[35] Jordan SC. Innate and adaptive immune responses to SARS-CoV-2 in humans: relevance to acquired immunity and vaccine responses. Clin Exp Immunol. 2021;204(3):310-320. doi:10.1111/cei.13582
[36] Ricci D, Etna MP, Rizzo F, Sandini S, Severa M, Coccia EM. Innate Immune Response to SARS- CoV-2 Infection: From Cells to Soluble Mediators. Int J Mol Sci. 2021;22(13):7017. Published 2021 Jun 29. doi:10.3390/ijms22137017
[37] Zhou Y, Fu B, Zheng X, et al. Pathogenic T-cells and inflammatory monocytes incite inflammatory storms in severe COVID-19 patients. Natl Sci Rev. 2020;7(6):998-1002. doi:10.1093/nsr/nwaa041
[38] Boechat JL, Chora I, Morais A, Delgado L. The immune response to SARS-CoV-2 and COVID- 19 immunopathology - Current perspectives. Pulmonology. 2021;27(5):423-437. doi:10.1016/j.pulmoe.2021.03.008
[39] Copaescu A, Smibert O, Gibson A, Phillips EJ, Trubiano JA. The role of IL-6 and other mediators in the cytokine storm associated with SARS-CoV-2 infection. J Allergy Clin Immunol. 2020;146(3):518-534.e1. doi:10.1016/j.jaci.2020.07.001
[40] Carvelli J, Demaria O, Vély F, et al. Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis. Nature. 2020;588(7836):146-150. doi:10.1038/s41586-020-2600-6
[41] Lei X, Dong X, Ma R, et al. Activation and evasion of type I interferon responses by SARS-CoV- 2. Nat Commun. 2020;11(1):3810. Published 2020 Jul 30. doi:10.1038/s41467-020-17665-9
[42] COVID-19-vaccines[EB/OL].
[2022/3/3].https://www.who.int/publications/m/item/draft- landscape-of-covid-19-candidate-vaccines
[43] Gresset-Bourgeois V, Leventhal PS, Pepin S, et al. Quadrivalent inactivated influenza vaccine (VaxigripTetra™). Expert Rev Vaccines. 2018;17(1):1-11. doi:10.1080/14760584.2018.1407650
[44] Wang H, Zhang Y, Huang B, et al. Development of an Inactivated Vaccine Candidate, BBIBP- CorV, with Potent Protection against SARS-CoV-2. Cell. 2020;182(3):713-721.e9. doi:10.1016/j.cell.2020.06.008
[45] Wu Z, Hu Y, Xu M, et al. Safety, tolerability, and immunogenicity of an inactivated SARS-CoV- 2 vaccine (CoronaVac) in healthy adults aged 60 years and older: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial. Lancet Infect Dis. 2021;21(6):803-812. doi:10.1016/S1473-3099(20)30987-7
[46] Coelingh K, Olajide IR, MacDonald P, Yogev R. Efficacy and effectiveness of live attenuated influenza vaccine in school-age children. Expert Rev Vaccines. 2015;14(10):1331-1346. doi:10.1586/14760584.2015.1078732
[47] Lundstrom K. Application of Viral Vectors for Vaccine Development with a Special Emphasis on COVID-19. Viruses. 2020;12(11):1324. Published 2020 Nov 18. doi:10.3390/v12111324
[48] Ledgerwood JE, DeZure AD, Stanley DA, et al. Chimpanzee Adenovirus Vector Ebola Vaccine. N Engl J Med. 2017;376(10):928-938. doi:10.1056/NEJMoa1410863
[49] Zhu FC, Guan XH, Li YH, et al. Immunogenicity and safety of a recombinant adenovirus type-5- vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet. 2020;396(10249):479-488. doi:10.1016/S0140- 6736(20)31605-6
[50] Sakurai F, Tachibana M, Mizuguchi H. Adenovirus vector-based vaccine for infectious diseases. Drug Metab Pharmacokinet. 2022;42:100432. doi:10.1016/j.dmpk.2021.100432
[51] Logunov DY, Dolzhikova IV, Zubkova OV, et al. Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia [published correction appears in Lancet. 2021 Jan 9;397(10269):98]. Lancet. 2020;396(10255):887-897. doi:10.1016/S0140-6736(20)31866-3
[52] Sadoff J, Le Gars M, Shukarev G, et al. Interim Results of a Phase 1-2a Trial of Ad26.COV2.S Covid-19 Vaccine. N Engl J Med. 2021;384(19):1824-1835. doi:10.1056/NEJMoa2034201
[53] Folegatti PM, Ewer KJ, Aley PK, et al. Safety and immunogenicity of the ChAdOx1 nCoV-19vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial [published correction appears in Lancet. 2020 Aug 15;396(10249):466] [published correction appears in Lancet. 2020 Dec 12;396(10266):1884]. Lancet. 2020;396(10249):467-478. doi:10.1016/S0140-6736(20)31604-4
[54] Zhu FC, Li YH, Guan XH, et al. Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial. Lancet. 2020;395(10240):1845-1854. doi:10.1016/S0140-6736(20)31208-3
[55] 张 佳 星 . 首 个 国 产 重 组 新 冠 病 毒 蛋 白 疫 苗 “ 转 正 ” 获 批 [N]. 科 技 日 报 ,2022-03- 04(003).DOI:10.28502/n.cnki.nkjrb.2022.001081.
[56] Yang S, Li Y, Dai L, et al. Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: two randomised, double-blind, placebo-controlled, phase 1 and 2 trials. Lancet Infect Dis. 2021;21(8):1107-1119. doi:10.1016/S1473-3099(21)00127-4
[57] Richmond P, Hatchuel L, Dong M, et al. Safety and immunogenicity of S-Trimer (SCB-2019), a protein subunit vaccine candidate for COVID-19 in healthy adults: a phase 1, randomised, double-blind, placebo-controlled trial. Lancet. 2021;397(10275):682-694. doi:10.1016/S0140- 6736(21)00241-5
[58] Roldão A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM. Virus-like particles in vaccine development. Expert Rev Vaccines. 2010;9(10):1149-1176. doi:10.1586/erv.10.115
[59] Silveira MM, Moreira GMSG, Mendonça M. DNA vaccines against COVID-19: Perspectives and challenges. Life Sci. 2021;267:118919. doi:10.1016/j.lfs.2020.118919
[60] Deering RP, Kommareddy S, Ulmer JB, Brito LA, Geall AJ. Nucleic acid vaccines: prospects for non-viral delivery of mRNA vaccines. Expert Opin Drug Deliv. 2014;11(6):885-899. doi:10.1517/17425247.2014.901308
[61] Lamb YN. BNT162b2 mRNA COVID-19 Vaccine: First Approval. Drugs. 2021;81(4):495-501. doi:10.1007/s40265-021-01480-7
[62] Li L, Petrovsky N. Molecular mechanisms for enhanced DNA vaccine immunogenicity. Expert Rev Vaccines. 2016;15(3):313-329. doi:10.1586/14760584.2016.1124762
[63] 金翔,俞庆龄,张璐楠,何悦,程鑫,刘晓雁,王宾.针对新型冠状病毒的 DNA 疫苗研究进展[J].中国 新药杂志,2020,29(21):2425-2433.
[64] Pfeffer M, Wiedmann M, Batt CA. Applications of DNA amplification techniques in veterinary diagnostics. Vet Res Commun. 1995;19(5):375-407. doi:10.1007/BF01839319
[65] Dey A, Chozhavel Rajanathan TM, Chandra H, et al. Immunogenic potential of DNA vaccine candidate, ZyCoV-D against SARS-CoV-2 in animal models. Vaccine. 2021;39(30):4108-4116. doi:10.1016/j.vaccine.2021.05.098
[66] COVID-19-vaccines[EB/OL].
[2021/9/2]. https://www.nature.com/articles/d41586-021-02385-x
[67] Momin T, Kansagra K, Patel H, et al. Safety and Immunogenicity of a DNA SARS-CoV-2 vaccine (ZyCoV-D): Results of an open-label, non-randomized phase I part of phase I/II clinicalstudy by intradermal route in healthy subjects in India. EClinicalMedicine. 2021;38:101020. doi:10.1016/j.eclinm.2021.101020
[68] 艾棣维欣(苏州)生物制药有限公司.新型冠状病毒 DNA 疫苗:中国,202111090409 .3(专利申 请号)2021-09-17
[69] Tebas P, Yang S, Boyer JD, et al. Safety and immunogenicity of INO-4800 DNA vaccine against SARS-CoV-2: A preliminary report of an open-label, Phase 1 clinical trial. EClinicalMedicine. 2021;31:100689. doi:10.1016/j.eclinm.2020.100689
[70] Andrade VM, Christensen-Quick A, Agnes J, et al. INO-4800 DNA vaccine induces neutralizing antibodies and T cell activity against global SARS-CoV-2 variants. NPJ Vaccines. 2021;6(1):121. Published 2021 Oct 14. doi:10.1038/s41541-021-00384-7
[71] Kraynyak KA, Blackwood E, Agnes J, et al. SARS-CoV-2 DNA Vaccine INO-4800 Induces Durable Immune Responses Capable of Being Boosted in a Phase 1 Open-Label Trial [published online ahead of print, 2022 Jan 25]. J Infect Dis. 2022;jiac016. doi:10.1093/infdis/jiac016
[72] Ahn JY, Lee J, Suh YS, et al. Safety and immunogenicity of two recombinant DNA COVID-19 vaccines containing the coding regions of the spike or spike and nucleocapsid proteins: an interim analysis of two open-label, non-randomised, phase 1 trials in healthy adults. Lancet Microbe. 2022;3(3):e173-e183. doi:10.1016/S2666-5247(21)00358-X
[73] Anderson EJ, Rouphael NG, Widge AT, et al. Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. N Engl J Med. 2020;383(25):2427-2438. doi:10.1056/NEJMoa2028436
[74] Hsieh CL, Goldsmith JA, Schaub JM, et al. Structure-based design of prefusion-stabilized SARS- CoV-2 spikes. Science. 2020;369(6510):1501-1505. doi:10.1126/science.abd0826

所在学位评定分委会
医学院
国内图书分类号
Q939.91
来源库
人工提交
成果类型学位论文
条目标识符http://sustech.caswiz.com/handle/2SGJ60CL/342750
专题南方科技大学医学院
推荐引用方式
GB/T 7714
候孟奇. 新型冠状病毒DNA疫苗的免疫原性和保护效果研究[D]. 深圳. 南方科技大学,2022.
条目包含的文件
文件名称/大小 文献类型 版本类型 开放类型 使用许可 操作
11930141-候孟奇-南方科技大学医(2510KB)----限制开放--请求全文
个性服务
原文链接
推荐该条目
保存到收藏夹
查看访问统计
导出为Endnote文件
导出为Excel格式
导出为Csv格式
Altmetrics Score
谷歌学术
谷歌学术中相似的文章
[候孟奇]的文章
百度学术
百度学术中相似的文章
[候孟奇]的文章
必应学术
必应学术中相似的文章
[候孟奇]的文章
相关权益政策
暂无数据
收藏/分享
所有评论 (0)
[发表评论/异议/意见]
暂无评论

除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。