WO2023159787A1 - Bcg基因bcg_1820在制备结核疫苗重组bcg中的应用 - Google Patents

Bcg基因bcg_1820在制备结核疫苗重组bcg中的应用 Download PDF

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WO2023159787A1
WO2023159787A1 PCT/CN2022/095396 CN2022095396W WO2023159787A1 WO 2023159787 A1 WO2023159787 A1 WO 2023159787A1 CN 2022095396 W CN2022095396 W CN 2022095396W WO 2023159787 A1 WO2023159787 A1 WO 2023159787A1
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bcg
gene
tuberculosis
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recombinant
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戈宝学
王琳
彭程
杨华
王洁
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上海市肺科医院
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  • the invention relates to the field of biomedicine, in particular to the application of BCG gene BCG_1820 in preparing recombinant BCG of tuberculosis vaccine.
  • Tuberculosis is the leading cause of death from bacterial infectious diseases worldwide.
  • the global tuberculosis situation remains grim due to the lack of an effective preventive vaccine against tuberculosis in adults.
  • BCG Mycobacterium bovisbacille Calmette-Guérin
  • BCG was first administered to newborns in Paris in 1921 1 .
  • BCG has a protective effect of more than 70% against tuberculous meningitis in newborns and school-aged children 3 .
  • the protective effect against tuberculosis in adults was very limited.
  • the development of new tuberculosis vaccines follows two basic pathways6,7.
  • the first approach is to replace BCG with modified recombinant BCG (rBCG) or gene knockout attenuated M. tuberculosis.
  • Genetically modified rBCG should be characterized as: a) safer; b) more immunogenic; c) induce longer-lasting protection; d) protect against highly virulent clinical isolates, such as Mycobacterium tuberculosis Beijing strain, Multidrug-resistant strains (MDR), extensively drug-resistant TB strains (XDR), etc.
  • One method of recombinant BCG is to introduce immunogenic tuberculosis-specific antigens that are lacking in BCG, such as the tuberculosis antigen gene encoded by RD1 (ESAT6, CFP10); or by overexpressing BCG autoantigens (homologues of the Ag85 complex, etc. ).
  • Another approach to recombinant BCG is gene editing of existing BCG to better enhance the host's natural immune response8 .
  • another strategy for developing a TB vaccine is to attenuate M. tuberculosis. This includes deletion of essential metabolic genes to generate auxotrophic mutants, or major deletion of virulence genes and their regulators.
  • subunit vaccines are non-live vaccines, or non-replicating vaccines that use viruses as vectors.
  • Subunit vaccines for tuberculosis are mainly recombinant proteins, or use attenuated viral vectors.
  • subunit vaccines can theoretically be used as priming vaccines, the prevailing view is that they can only be used as booster vaccines on top of adjuvant BCG, recombinant BCG, or attenuated Mtb vaccines.
  • BCG BCG BCG has limited protective effect on tuberculosis patients, and the construction of recombinant BCG is the main research direction.
  • the main research strategy of recombinant BCG is to knock out the virulence gene of BCG, activate the immune response function of the host, and improve the protective effect of the existing vaccine BCG.
  • which genes are the important immunosuppressive factors of BCG is still unclear, and there is still a lack of theoretical basis for gene editing of BCG based on which target can improve the protective effect of BCG.
  • Antimicrobial peptides are considered to be the ancient defense weapons of the innate immune system of organisms, and have a wide range of activities against Gram-positive and Gram-negative bacteria, fungi, parasites and viruses.
  • AMPs are usually composed of 12-15 amino acids with cations (consisting of positively charged arginine and lysine residues) 10 , and their mechanism of action is to form interactions with negatively charged bacterial membranes, thereby producing Phospholipid replacement, membrane disorder and internalization 11 . Because AMPs have different mechanisms of action, microbes rarely develop resistance.
  • the invention searches for the BCG virulence gene that significantly inhibits its expression, and provides a strategy for constructing a recombinant BCG with better effect.
  • the present invention found that the recombinant BCG strain constructed by deleting the virulence gene BCG_1820 on the wild-type BCG strain can significantly improve the immune protection effect of BCG, and provide candidates for the development of tuberculosis vaccines. Based on this, the present invention provides The application of BCG gene BCG_1820 in the preparation of recombinant BCG for tuberculosis vaccine was investigated.
  • the present invention adopts the following technical solutions:
  • the first aspect of the present invention is to provide a BCG recombinant bacterium ⁇ BCG_1820, in which the gene BCG_1820 is knocked out.
  • the BCG_1820 gene gRNA sequence used is SEQ ID No.:2.
  • the second aspect of the present invention is to provide a method for constructing the above-mentioned BCG recombinant bacteria, which is obtained by knocking out the gene BCG_1820 in the wild-type BCG strain by CRISPR/Cas9 technology.
  • the third aspect of the present invention is to provide the application of the above-mentioned BCG recombinant bacteria in the preparation of tuberculosis vaccine, and the tuberculosis vaccine contains the BCG recombinant bacteria.
  • tuberculosis vaccine also includes an adjuvant.
  • the fourth aspect of the present invention is to provide a recombinant BCG tuberculosis vaccine, which is BCG with gene BCG_1820 knocked out.
  • BCG_1820 gene was knocked out by CRISPR/Cas9 technology.
  • the BCG_1820 gene gRNA sequence used is SEQ ID No.:2.
  • the fifth aspect of the present invention is to provide a BCG strain BCG_1820 gene knockout vector, which is a gRNA expression vector based on the CRISPR/Cas9 system, and the gRNA sequence is SEQ ID No.:2.
  • the sixth aspect of the present invention is to provide the application of the above-mentioned BCG strain BCG_1820 gene knockout vector in the preparation of tuberculosis vaccine.
  • the present invention adopts the above technical scheme, and compared with the prior art, it has the following technical effects:
  • the BCG recombinant bacterium ⁇ BCG_1820 provided by the present invention can obviously induce macrophages to produce more antibacterial peptides, give the host a stronger ability to resist tuberculosis infection, and has the potential to be a candidate vaccine for tuberculosis.
  • Figure 1 shows the results of PCR identification of BCG_1820 gene knockout strains in an embodiment of the present invention
  • Figure 2 shows that the ⁇ BCG_1820 strain significantly promotes the expression of macrophage antimicrobial peptide genes in an embodiment of the present invention; among them, Figures A-D respectively show the effects of the ⁇ BCG_1820 strain on the expression levels of Camp, Hamp, Defb3 and Defb4 in macrophages;
  • Fig. 3 is the flowchart of mouse immune challenge experiment in one embodiment of the present invention.
  • Fig. 4 has shown the comparison result of the lung tissue load of bacteria 30 days after the mouse immunization 30 days in an embodiment of the present invention
  • Figure 5 shows the HE staining results (Figure A) and acid-fast staining results (Figure B) of lung pathology of immunized mice 30 days after infection in an embodiment of the present invention.
  • the present invention provides the application of BCG gene BCG_1820 in preparing recombinant BCG of tuberculosis vaccine, wherein the amino acid sequence of gene BCG_1820 (source database: https://www.uniprot.org/uniprot/A0A0H3M6W4) is SEQ ID No.:1.
  • the methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified.
  • a BCG_1820 gene deletion strain was constructed on the wild-type BCG strain.
  • the specific construction process and results are as follows:
  • a BCG_1820 gene deletion strain ( ⁇ BCG_1820) was constructed using Cas9 technology on the wild-type BCG Danish strain.
  • the sgRNA expression plasmids of Cas9 and BCG_1820 gene were electroporated (wherein, the gRNA sequence of BCG_1820 gene is ATCGGCTCCGCATTGAACGC (SEQ ID No.: 2)), spread on K+Zeo resistant culture plate after amplification, and pick After the single clone was taken, it was identified by PCR and sequencing, and the results are shown in Figure 1.
  • Example 1 On the basis of Example 1, it is verified that the ⁇ BCG_1820 strain can induce macrophages to produce more antimicrobial peptides.
  • the specific experimental steps and results are as follows:
  • BCG can significantly promote the expression of antimicrobial peptides after knocking out the BCG_1820 gene, which suggests that the protein encoded by the BCG_1820 gene can inhibit the expression of host antimicrobial peptides and is a virulence factor of BCG.
  • mice were injected with PBS, 1 ⁇ 10 6 CFU of BCG strain, or 1 ⁇ 10 6 CFU of ⁇ BCG_1820 strain through tail vein respectively.
  • the mice in each group were given respiratory tract infection of Mycobacterium tuberculosis H37Rv strain in a biosafety level 3 laboratory.
  • the mice were sacrificed by neck dislocation, and the lung tissues were separated for CFU counting to confirm the amount of bacteria in the lung tissues of the mice in each group.
  • the lung tissues of the mice in each group were fixed with 4% PFA and passed through paraffin. Embedding, tissue section and H&E staining were used to observe the pathological changes of lung tissue in each group.
  • the ⁇ BCG_1820 strain can better protect the host against tuberculosis infection than the BCG strain.

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Abstract

提供了一种BCG_1820基因被敲除的BCG重组菌ΔBCG_1820、其制备方法，及其在制备结核疫苗中的应用。所述BCG重组菌ΔBCG_1820可以诱导巨噬细胞产生更多的抗菌肽，给予宿主更强的抵抗结核菌感染的能力，有潜力作为结核菌的候选疫苗。

Description

BCG基因BCG_1820在制备结核疫苗重组BCG中的应用 技术领域
本发明涉及生物医药领域，具体涉及BCG基因BCG_1820在制备结核疫苗重组BCG中的应用。
背景技术
结核病在世界范围是细菌性传染病的主要死亡原因。由于缺乏对成年人肺结核有效的预防性疫苗，全球结核病形势依然严峻。目前唯一获得临床使用许可用于预防结核病的疫苗是卡介苗(Mycobacterium bovisbacille Calmette-Guérin，BCG)，这是一种减毒活疫苗。卡介苗于1921年在巴黎首次给新生儿注射 1。到2019年，全球88％的儿童在出生后第一年接受卡介苗接种 2。卡介苗对新生儿和学龄儿童的结核性脑膜炎具有70％以上的保护作用 3。然而，对成年人肺结核的保护效果非常有限。21世纪初，全球新型结核病疫苗的研发力度不断加大。这些包括Mtb融合蛋白的亚单位佐剂制剂、表达Mtb一种或多种抗原的病毒载体疫苗、灭活分枝杆菌疫苗和减毒分枝杆菌疫苗 4,5。
新结核病疫苗的开发遵循两条基本途径 6,7。第一种途径是用改良的重组卡介苗(rBCG)或基因敲除减毒的结核分枝杆菌取代卡介苗。基因改良rBCG的特点应该是：a)更安全；b)免疫原性更强；c)诱导更持久的保护；d)对高毒力临床分离株具有保护作用，如结核分枝杆菌北京株、多耐药菌株(MDR)、广泛耐药结核病菌株(XDR)等。重组卡介苗的一种方法是引入卡介苗中缺乏的免疫原性结核菌特异性抗原，如RD1编码的结核抗原基因(ESAT6，CFP10)；或者通过过表达卡介苗自身抗原(Ag85复合物的同源物等)。另一种重组卡介苗的方法是对现有卡介苗进行基因编辑，以更好地增强宿主天然免疫反应 8。除了这两种rBCG疫苗方法外，另外一种开发结核疫苗的策略是对结核分枝杆菌进行减毒。这包括删除必需的代谢基因以产生营养缺陷型突变体，或主要缺失毒力基因及其调控因子。一项研究表明在田鼠分枝杆菌上表达结核分枝杆菌的RD1抗原显著提高了宿主对结核菌感染的抵抗能力 9。还有研究发现重组耻垢分枝杆菌也 可以作为结核疫苗。当耻垢分枝杆菌敲除esx-3基因时，可以看到免疫小鼠出现强烈的先天免疫反应。当这种重组耻垢分枝杆菌回转入结核菌的esx-3基因时，在小鼠结核菌攻毒模型中观察到对宿主更好的保护活性。
开发结核病疫苗的第二个主要途径是构建亚单位疫苗。这些疫苗是非活体疫苗，或者以病毒为载体的非复制疫苗。结核病的亚单位疫苗主要是重组蛋白，或使用减毒病毒载体。虽然亚单位疫苗理论上可以用作启动疫苗，但目前主流观点是，它们只能用作辅助卡介苗、重组卡介苗或减毒Mtb疫苗之上的增强疫苗。
目前临床使用的BCG卡介苗对肺结核患者的保护效果有限，构建重组卡介苗是主要研究方向。重组卡介苗的主要研究策略是敲除BCG的毒力基因，激活宿主的免疫应答功能，提高现有疫苗BCG的保护效果。但是哪些基因是BCG重要的免疫抑制因子尚不明确，以哪个靶点为基础对BCG进行基因编辑可以提高BCG的保护效果还缺少理论基础。
抗菌肽(Antimicrobial Peptides，AMPs)被认为是生物体先天免疫系统的古老防御武器，具有广泛的对抗革兰氏阳性和革兰氏阴性细菌、真菌、寄生虫和病毒的活性。AMPs通常由12-15个氨基酸组成，带有阳离子(由带正电的精氨酸和赖氨酸残基组成) 10，其作用机制的是与带负电的菌体膜形成相互作用，从而产生磷脂置换、膜结构紊乱和内部化 11。由于AMPs的作用机制不同，因此微生物很少产生耐药性。本发明基于宿主的抗菌肽，寻找显著抑制其表达的BCG毒力基因，为构建效果更好的重组卡介苗提供策略。
发明内容
为了克服现有技术中的缺陷，本发明发现在野生型BCG菌株上缺失毒力基因BCG_1820构建的重组BCG菌株以显著提高BCG的免疫保护效果，为结核疫苗开发提供候选，基于此，本发明提供了BCG基因BCG_1820在制备结核疫苗重组BCG中的应用。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面是提供一种BCG重组菌ΔBCG_1820，该BCG重组菌中的基因BCG_1820被敲除。
进一步地，该BCG重组菌中的基因BCG_1820通过CRISPR/Cas9技术被敲 除。
进一步地，在敲除过程中，采用的BCG_1820基因gRNA序列为SEQ ID No.:2。
本发明的第二方面是提供上述BCG重组菌的构建方法，通过CRISPR/Cas9技术敲除野生型BCG菌株中的基因BCG_1820获得该BCG重组菌。
本发明的第三方面是提供上述BCG重组菌在制备结核疫苗中的应用，该结核疫苗包含该BCG重组菌。
进一步地，该结核疫苗还包括佐剂。
本发明的第四方面是提供一种结核疫苗重组BCG，为基因BCG_1820被敲除的卡介苗。
进一步地，BCG_1820基因通过CRISPR/Cas9技术被敲除。
进一步地，在敲除过程中，采用的BCG_1820基因gRNA序列为SEQ ID No.:2。
本发明的第五方面是提供一种BCG菌株BCG_1820基因敲除载体，该敲除载体是基于CRISPR/Cas9系统的gRNA表达载体，所述gRNA序列为SEQ ID No.:2。
本发明的第六方面是提供上述BCG菌株BCG_1820基因敲除载体在制备结核疫苗中的应用。
本发明采用以上技术方案，与现有技术相比，具有如下技术效果：
本发明提供的BCG重组菌ΔBCG_1820可以明显诱导巨噬细胞产生更多的抗菌肽，给予宿主更强的抵抗结核菌感染的能力，有潜力作为结核菌的候选疫苗。
附图说明
图1显示了本发明一实施例中PCR鉴定BCG_1820基因敲除菌株的结果；
图2显示了本发明一实施例中ΔBCG_1820菌株显著促进巨噬细胞抗菌肽基因表达；其中，图A-D分别显示了ΔBCG_1820菌株对巨噬细胞中Camp，Hamp，Defb3和Defb4表达量的影响；
图3是本发明一实施例中小鼠免疫攻毒实验的流程图；
图4显示了本发明一实施例中小鼠免疫接种30天后攻毒30天肺组织荷菌量 的比较结果；
图5显示了本发明一实施例中免疫接种小鼠感染30天后肺部病理HE染色结果(图A)和抗酸染色结果(图B)。
具体实施方式
本发明提供了BCG基因BCG_1820在制备结核疫苗重组BCG中的应用，其中，基因BCG_1820(来源数据库：https://www.uniprot.org/uniprot/A0A0H3M6W4)的氨基酸序列为SEQ ID No.:1。
下面通过具体实施例和附图对本发明进行详细和具体的介绍，以使更好的理解本发明，但是下述实施例并不限制本发明范围。
实施例中方法如无特殊说明的采用常规方法，使用的试剂如无特殊说明的使用常规市售试剂或按常规方法配制的试剂。
实施例1
本实施在野生型BCG菌株上构建了BCG_1820基因缺失菌株，具体的构建过程和结果如下：
在野生型BCG丹麦菌株上利用Cas9技术构建BCG_1820基因缺失菌株(ΔBCG_1820)。
首先制备制BCG:pYC1759的感受态细胞，将pYC1759质粒电转入野生型的BCG丹麦菌株，挑取成功转入质粒的BCG单克隆菌株后在K+的7H9+OADC培养基中进行菌体扩增，随后进行甘油水洗三次，收集感受态细胞-80度冻存备用。在BCG:pYC1759感受态细胞中电转Cas9以及BCG_1820基因的sgRNA表达质粒(其中，BCG_1820基因gRNA序列为ATCGGCTCCGCATTGAACGC(SEQ ID No.:2))，扩增后涂于K+Zeo抗性培养平板，挑取单克隆后进行PCR及测序鉴定，结果如图1所示。
实施例2
本实施例在实施例1的基础上，验证ΔBCG_1820菌株可以诱导巨噬细胞产生更多的抗菌肽，具体的实验步骤和结果如下：
利用小鼠腹腔原代巨噬细胞感染模型，感染野生型BCG菌株和ΔBCG_1820菌株(MOI＝5)12小时、24小时后Trizol裂解细胞，抽提总RNA，反转录为cDNA 后通过QPCR对细胞内的Camp，Hamp，Defb3和Defb4进行定量分析。
如图2所示，BCG敲除BCG_1820基因后可以显著促进抗菌肽的表达，这提示BCG_1820基因编码的蛋白可以抑制宿主抗菌肽表达，是BCG的毒力因子。
实施例3
本实施例在动物水平上验证ΔBCG_1820菌株比BCG菌株具有更强的免疫保护功能，具体的实验步骤和结果如下：
参考图3的流程图，分别给予野生型C57BL/6小鼠尾静脉注射PBS、1×10 6CFU的BCG菌株，或者1×10 6CFU的ΔBCG_1820菌株。免疫30天后在生物安全三级实验室中给予各组小鼠经呼吸道感染结核菌H37Rv菌株。感染结核菌30天后颈部脱臼处死小鼠，分离肺组织进行CFU计数，确认各组小鼠肺组织内的荷菌量，同时利用4％的PFA对各组小鼠的肺组织进行固定通过石蜡包埋，组织切片及H&E染色观察各组间肺组织病理改变。
如图4所示，ΔBCG_1820菌株相比免疫野生型BCG菌株肺组织和菌量下降了30倍，同时可见更少的中性粒细胞浸润和更多的完好的肺泡组织(如图5)。
综上所述，ΔBCG_1820菌株比BCG菌株可以更好的保护宿主抵抗结核菌感染。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
参考文献：
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Claims (10)

  1. 一种BCG重组菌，其特征在于，为ΔBCG_1820菌株，所述BCG重组菌中的基因BCG_1820被敲除。
  2. 根据权利要求1所述的BCG重组菌，其特征在于，所述BCG重组菌中的基因BCG_1820通过CRISPR/Cas9技术被敲除。
  3. 根据权利要求2所述的BCG重组菌，其特征在于，在敲除过程中，采用的BCG_1820基因gRNA序列为SEQ ID No.:2。
  4. 如权利要求1-3任一项所述的BCG重组菌的构建方法，其特征在于，通过CRISPR/Cas9技术敲除野生型BCG菌株中的基因BCG_1820获得所述BCG重组菌。
  5. 如权利要求1-3任一项所述的BCG重组菌在制备结核疫苗中的应用，其特征在于，所述结核疫苗包含所述BCG重组菌。
  6. 根据权利要求6所述的应用，其特征在于，所述结核疫苗还包括佐剂。
  7. 一种结核疫苗重组BCG，其特征在于，为基因BCG_1820被敲除的卡介苗。
  8. 根据权利要求7所述的结核疫苗重组BCG，其特征在于，BCG_1820基因通过CRISPR/Cas9技术被敲除；在敲除过程中，采用的BCG_1820基因gRNA序列优选为SEQ ID No.:2。
  9. 一种BCG菌株BCG_1820基因敲除载体，其特征在于，所述敲除载体是基于CRISPR/Cas9系统的gRNA表达载体，所述gRNA序列为SEQ ID No.:2。
  10. 如权利要求9所述的BCG菌株BCG_1820基因敲除载体在制备结核疫苗中 的应用。
PCT/CN2022/095396 2022-02-24 2022-05-26 Bcg基因bcg_1820在制备结核疫苗重组bcg中的应用 WO2023159787A1 (zh)

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