CN111249308A - Car-t技术在预防和治疗冠状病毒感染性疾病中的应用 - Google Patents
Car-t技术在预防和治疗冠状病毒感染性疾病中的应用 Download PDFInfo
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Abstract
本发明属于T细胞免疫治疗领域，具体涉及CAR‑T技术在预防和治疗冠状病毒感染性疾病中的应用。冠状病毒感染性疾病为SARS‑CoV或nCoV‑19或MERS‑CoV或HCoV‑229E或HCoV‑OC43或HCoV‑NL63或HCoV‑HKU1感染引起的疾病。该疗法效果好，尤其适用于重症感染的应急治疗，受病毒变异影响小，可应用于大范围预防免疫。
Description
技术领域
本发明属于T细胞免疫治疗领域，具体涉及CAR-T技术在预防和治疗冠状病毒感染中的应用。
发明背景
迄今为止，目前共发现7种可感染人类的冠状病毒，其中，中东呼吸综合征冠状病毒(MERS-CoV)、严重急性呼吸综合征冠状病毒(SARS-CoV)和2019新型冠状病毒(nCoV-19)所致疾病的严重程度以及社会影响更大。其余是HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1。
感染nCoV-19、SARS和MERS病毒，会导致严重的肺炎，以发热为首发症状，偶有畏寒，同时伴有头痛、关节酸痛和全身酸痛、乏力。另外有明显的呼吸道症状：干咳、少痰，个别病人偶有血丝痰，部分病人出现呼吸加速、气促等上呼吸道病毒感染症状，严重引起呼吸衰竭死亡。
冠状病毒多在上皮组织中生长，主要通过鼻、眼、呼吸道粘膜感染，可以通过皮下注射感染。冠状病毒的抗原变异性高，存在有多种血清型并有抗原的变异，导致疫苗和特异性抗体研究困难重重，已经接种疫苗的人群可以发生重复感染。针对不断变异的野生病毒，目前尚无特效预防和治疗手段。
冠状病毒基因组序列具有同源性，可分为4个属，α，β，γ和δ。γ和δ冠状病毒属只感染动物，α冠状病毒属包括人冠状病毒29E和NL63，β冠状病毒属包括4个家系，HKU1和OC43属于A家系；B家系包括SARS，与重症肺炎有关；MERS则属于β冠状病毒C家系。
冠状病毒属的病毒是具外套膜的正链单股RNA病毒。病毒包膜上存在棘突，不同的冠状病毒的棘突差异明显，看似冠状，所以命名为“冠状病毒”。病毒粒子外包着脂肪膜，膜表面有三种糖蛋白：刺突糖蛋白(S，SpikeProtein，是受体结合位点、溶细胞作用和主要抗原位点)；小包膜糖蛋白(E，EnvelopeProtein，较小，与包膜结合的蛋白)；膜糖蛋白(M，MembraneProtein，负责营养物质的跨膜运输、新生病毒出芽释放与病毒外包膜的形成)。少数种类还有血凝素糖蛋白(HE蛋白，Haemaglutinin-esterase)。冠状病毒的核酸为非节段单链(+)RNA，长27-31kd，是RNA病毒中最长的RNA核酸链，具有正链RNA特有的重要结构特征：即RNA链5’端有甲基化“帽子”，3’端有PolyA“尾巴”结构。这一结构与真核mRNA非常相似，也是其基因组RNA自身可以发挥翻译模板作用的重要结构基础，而省去了RNA-DNA-RNA的转录过程。冠状病毒的RNA和RNA之间重组率非常高，病毒极易出现变异。抗原性发生变化的结果是导致原有疫苗失效。
冠状病毒致病机制复杂，难以预防和治疗的主要机制有：主动改变宿主细胞状态以便于逃逸免疫清除、进而利于病毒本身复制和存活；2、病毒可以产生micrRNA诱导T抗原表达的减低使得感染细胞躲避T细胞。3、S蛋白是病毒和人体细胞受体结合的桥梁，冠状病毒S蛋白的多样性导致研制疫苗异常困难。4、易出现耐药株。
CAR-T(Chimeric Antigen Receptor T-Cell Immunotherapy，嵌合抗原受体T细胞免疫疗法)被认为是最有前景的免疫治疗方式之一。嵌合抗原受体(CAR)是CAR-T的核心部件，赋予T细胞HLA非依赖的方式识别抗原的能力，这使得经过CAR改造的T细胞相较于天然T细胞表面受体TCR能够识别更广泛的目标。
通过注射疫苗引发的抗体保护反应有效期都很有限。而针对保守的内源蛋白引发的T细胞免疫反应保护期则很长。T细胞包含多种亚型，其中细胞毒性T细胞(Tc)对病毒具有直接杀伤作用，辅助性T细胞(Th)协助B细胞产生病毒特异性抗体，调节性T细胞(Treg)调节病毒引起的异常免疫性损伤。记忆T细胞根据功能分为中心记忆T细胞(TCM)和效应记忆T细胞(TEM)。当CAR-T注入机体后，TEM数量显著增加。E/P-选择素+的TEM细胞具有迁移能力，能够进入周围组织。组织常驻T细胞(TRM)是一群特殊的TEM，感染结束后能长期存活于周围组织中。当同类病毒入侵时，TRM不依赖外周血记忆T细胞迅速清除病毒，对多种同类病毒感染都具有长期的保护作用。但目前TRM细胞还无法进行分离和培养，如何安全诱导机体大量产生TRM细胞预防病毒感染仍然是个难题。
病毒特异性CAR-T技术与病毒疫苗相比的优势：1、疫苗注射后逐渐产生抗体，CAR-T技术抗病毒作用快，可以根据不同人群需求调整剂量和给药途径，适用于重症患者的个体化应急治疗；2、针对冠状病毒保守膜抗原的CAR-TRM可以长期存在于鼻粘膜、眼结膜、呼吸道和皮肤中，能有效抵御多种同类病毒入侵，抗病毒作用更为广泛持久，适合于大规模人群预防免疫。
病毒进入组织细胞后，血液中的抗体便不能发挥作用，此时需要通过效应T细胞来杀伤细胞中的病毒。嵌合抗原抗体(CAR)可以通过基因和细胞工程技术制备，随着自动化的CAR-T细胞制备工艺涌入市场，治疗成本大幅下降。
CAR-T细胞可以诱导组织TRM细胞增多，TRM细胞能够长期存在于组织中，阻止多种同类病毒再次入侵感染。疫苗虽然也可以诱导机体产生TRM细胞，但细胞数量有限。针对冠状病毒极易变异的特点，皮下注射CAR-T适用于普通人群的基础免疫，弥补疫苗的不足。
CAR-T在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的免疫治疗方式之一，多次创造癌症治疗的奇迹。在非肿瘤性疾病中，CAR-T技术治疗研究最多的感染性疾病是艾滋病。迄今为止，还没有CAR-T技术用于治疗和预防冠状病毒感染的研究报道。
发明内容
本项目通过基因工程技术合成嵌合抗原受体，制备质粒电转T细胞分别获得特异性和保守性SARS-CoV膜蛋白CAR-T细胞；通过体外细胞培养显示CAR-T组分泌对病毒有杀伤作用的细胞因子IL-2和IFN-r，明显高于对照T细胞组。
分别通过小鼠尾静脉和皮下注射输入SARS-CoV特异性S膜蛋白CAR-T细胞，显示小鼠外周血中效应T细胞较对照组增多，具有抗病毒作用；同时显示SARS-CoV膜蛋白CAR-T能通过增加鼻上皮组织中TRM细胞，使机体获得对同类冠状病毒的长期免疫力，抵御病毒再次感染。
冠状病毒基因组序列具有同源性，冠状病毒属的病毒都是具外套膜的正链单股RNA病毒。病毒膜表面的一些膜糖蛋白在各种冠状病毒中具有高度的保守性。因此CAR-T对SARS-CoV的治疗和预防作用可以推广应用到其他冠状病毒感染的防治。
基于上述研究，本发明涉及了CAR-T疗法在治疗和预防冠状病毒感染性疾病中的应用，其特征在于所述冠状病毒感染性疾病为SARS-CoV或nCoV-19或MERS-CoV或HCoV-229E或HCoV-OC43或HCoV-NL63或HCoV-HKU1感染引起的疾病。优选地，所述CAR-T疗法在通过静脉滴注或者皮下注射治疗和预防冠状病毒感染性疾病中的应用；进一步优选地，CAR-T疗法在静脉滴注或者皮下注射治疗和预防SARS-CoV冠状病毒感染性疾病中的应用，所述CAR-T疗法在静脉滴注或者皮下注射治疗和预防nCoV-19冠状病毒感染性疾病中的应用；进一步地，所述CAR-T疗法在治疗和预防其他新发未知冠状病毒感染性疾病中的应用。
本发明与现有技术相比其有益效果是：本发明揭示了CAR-T技术治疗和预防冠状病毒感染性疾病的一种新用途，可以应用到其它新发未知冠状病毒感染性疾病的预防和治疗。该技术与抗体治疗相比，nCoV-19特异性抗原CAR-T技术对病毒变异反应更迅速，抗病毒作用快，适用于重症患者的个体化应急治疗。相比于疫苗，nCoV-19保守性抗原CAR-T抗病毒作用更为广泛持久，受病毒变异影响小，适合于大规模人群的预防免疫。此外，CAR-T技术治疗肿瘤和艾滋病主要是通过静脉治疗，已经发现细胞因子释放综合征、肝肾功能损伤等不良反应。如果可以通过皮下注射输入CAR-T，有望达到治疗更方便，不良反应更少的效果。
附图说明
图1 CAR-T细胞分泌细胞因子IL-2和IFN-r均高于T细胞组(P＜0.01)。
图2 CAR-T组(Model)小鼠皮肤中的表面抗原CD103、CD44和CD49a均显著性高于T细胞组(Control)(P＜0.01)。
具体实施方式
下面结合实施例对本发明做进一步说明，应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，凡在本发明的构思前提下对本发明制备方法的简单改进都属于本发明的保护范围之内。下面实施例未注明具体条件的实验方法，通常按照本领域的公知手段。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂公司购买得到的。
实施例1 SARS-CoV膜S蛋白受体CAR-T细胞的制备和杀伤能力研究
实验材料：
淋巴细胞分离液；无血清培养基；白介素2；细胞因子检测试剂盒；电转仪(德国Lonza公司)；流式细胞仪(美国BD公司)；聚合酶链反应(PCR)仪。
实验方法：
1、制备S膜蛋白受体CAR-T细胞
1.1靶向特异性膜抗原质粒的构建：基因库中调取SARS-CoV囊膜S蛋白基因序列，用连接PCR方法合成完整片段，测序确认后与人免疫球蛋白(IgG)序列Fc段连接，合成针对S膜蛋白的嵌合抗原受体(CAR)序列，并在两段分别加入酶切位点，并克隆至原核表达载体中。
1.2质粒电转至T细胞：
(1)采集抗凝全血：
从小鼠尾静脉采血，取一个洗涤剂瓶，中间(横向)剪开，瓶口段(纵向)剪开，即制作成了一个固定小鼠的简易器械，把小鼠放入洗涤剂瓶中进行操作。小鼠的尾静脉用手可以感觉到在尾部有一个狭缝，顺着狭缝进针取血。
(2)分离收集PBMC细胞：
将静脉血20ml用ACD抗凝剂以容积比血∶抗凝剂＝9∶1抗凝处理；按血∶分离液＝1∶2注入淋巴细胞分离液上层，400g 20℃离心30分钟；交接层重浮于4倍体积的RPMI1640并通过离心法200g，10min洗三次；获得细胞用于以下实验。
(3)质粒电转：
将0.1cm的电击杯放在冰上进行预冷，用100微升的细胞加入1微升纯化的连接液，或者加入0.5微升未纯化的连接液，小心混匀，在冰上放置二十分钟。将混合液加入预冷的电击杯中，注意擦干杯外的水，防止电火花，放入电转化仪的反应槽内，接上电源。然后，在3kv、200电阻和25微F的BioRad电转化仪，0.2电转化杯，时间常数在4.5-5ms之间，然后进行电击。在听到蜂鸣声后，向电击杯中迅速加入1ml 37℃的保温的SOC液体培养基，将混合液洗出，转移到1.5ml的离心管中。最后，在37℃220-250r/min复苏培养一小时，使其充分表达抗性，取适量涂平板，将平板倒置在培养箱中，37℃下培养14d，得到得到病毒特异性抗原受体CAR-T细胞(简称：特异抗原CAR-T)和病毒保守抗原受体CAR-T细胞(简称：保守抗原CAR-T)
2、S膜蛋白受体CAR-T杀伤病毒测试
在特异抗原CAR-T、保守抗原CAR-T、T细胞培养液中加入适量SARS病毒悬液，48h后收取上清。用细胞因子试剂盒检测IL-2，干扰素(IFN)-r含量。
实验结果：
1.流式检测结果：转染后T细胞表面SARS-CoV膜S蛋白CAR表达阳性率90％，表明CAR-T细胞构建成功。
2.SARS-CoV膜S蛋白CAR-T细胞分泌细胞因子情况：SARS-CoV膜S蛋白CAR-T细胞分泌的IL-2浓度为(216.06±15.03)ng/L，IFN-r浓度为(6835.8±231.26)ng/L，高于T细胞组分泌的IL-2(148.26±12.58)ng/L，IFN-r(2062.36±26.21)ng/L(图1)；差异有统计学意义(P＜0.05)。
实施例2小鼠体内研究CAR-T细胞治疗SARS病毒感染能力
材料：
SPF级6周龄BALB/c-nude小鼠；流式细胞仪(美国BD公司)；聚合酶链反应(PCR)仪。实验方法：
(1)购买6周龄雌性BALB/c-nude小鼠[(16±2)g]，无特定病原体(SPF)级动物中心培养1周适应环境后，随机分成4组(特异抗原CAR-T、保守抗原CAR-T、T细胞组、PBS组)。
(2)分别通过小鼠尾静脉、皮下注射特异抗原CAR-T、保守抗原CAR-T、T细胞、PBS。每3天记录小鼠体重和一般情况，采集静脉血。
(3)各组分别于第15和30天安乐死小鼠，收集鼻粘膜组织。
(4)T细胞亚型检测：通过PE/cy7标记的抗小鼠CD4抗体、PerCP-Cy5.5标记的抗小鼠CD8抗体、PE标记的抗小鼠CCR7抗体和Pacific Blue标记的抗小鼠CD45RA抗体(均购自美国Biolegend公司)标记T细胞，于流式细胞分析仪中检测不同亚型T细胞的比例。
(5)TRM细胞的检测：
分别取非感染部位皮肤、鼻粘膜以及肺组织，制备单细胞悬液，流式细胞仪检测CD3、CD69(H1.2F3)、CD103(M290)共表达T细胞数量。
具体操作步骤：把组织剪碎放在含有1mg/mL胶原酶A、40ug/mLDNase I的Hanks平衡盐溶液(HBSS)中37℃孵育30min。然后通过70um的尼龙细胞滤器，将细胞悬液离心(500g；10min；4℃)收集细胞，用冷的PBS清洗之后进行流式抗体染色30min。细胞重悬于100μL/管的含1％BSA的PBS染色缓冲液中并在4℃孵育15min，抗体溶度为1μg/100μL。染色完毕，离心(500g；10min；4℃)弃上清。用1mL1％BSA-PBS清洗细胞1次，离心(350g；4℃；5min)弃上清，重悬于300μL的1％BSA-PBS中，上机检测。
统计学方法：
应用SPSS 18.0统计软件分析，计量资料采用均值±标准差(Mean±SD)表示，组间比较采用t检验，多组间比较采用单因素方差分析，以P＜0.05为差异有统计学意义。
实验结果：
(1)CAR-T细胞组和T细胞组中CD4+和CD8+T细胞比例差异无统计学意义(CD4+T细胞比例：41％对37％，P＞0.05；CD8+T细胞比例：52％对57％，P＞0.05)。以CCR7和CD45RA为表面标记，检测和分析CAR-T细胞组和T细胞组中记忆性T细胞亚群(Tem、Tem、Temra和naive T细胞)的比例。结果显示，与T细胞相比，CAR-T细胞组的效应性记忆T细胞(Tem，CCR7-CD45RA-)比例较T细胞组高(P＜0.01)(58.6％对32.9％)。
(2)CAR-T组小鼠皮肤中的表面抗原CD103、CD44和CD49a均显著性高于T细胞组(P＜0.01)(图2)。
Claims (6)
1.CAR-T疗法在治疗和预防冠状病毒感染性疾病中的应用，其特征在于所述CAR-T疗法属于T细胞免疫治疗领域，其特征在于所述冠状病毒为SARS-CoV或nCoV-19或MERS-CoV或HCoV-229E或HCoV-OC43或HCoV-NL63或HCoV-HKU1；其特征在于所述冠状病毒感染性疾病为SARS-CoV或nCoV-19或MERS-CoV或HCoV-229E或HCoV-OC43或HCoV-NL63或HCoV-HKU1感染引起的疾病。
2.根据权利要求1所述CAR-T疗法在治疗和预防冠状病毒感染性疾病中的应用，其特征在于所述CAR-T的抗原是冠状病毒膜蛋白。
3.根据权利要求1所述CAR-T疗法在治疗和预防冠状病毒感染性疾病中的应用，其特征在于所述CAR-T疗法在通过静脉滴注或者皮下注射治疗和预防冠状病毒感染性疾病中的应用。
4.根据权利要求2所述CAR-T疗法在治疗和预防冠状病毒感染性疾病中的应用，其特征在于所述CAR-T疗法在静脉滴注或者皮下注射治疗和预防SARS-CoV冠状病毒感染性疾病中的应用。
5.根据权利要求2所述CAR-T疗法在治疗和预防冠状病毒感染性疾病中的应用，其特征在于所述CAR-T疗法在静脉滴注或者皮下注射治疗和预防nCoV-19冠状病毒感染性疾病中的应用。
6.根据权利要求2所述CAR-T疗法在治疗和预防冠状病毒感染性疾病中的应用，其特征在于所述CAR-T疗法在静脉滴注或者皮下注射治疗和预防MERS-CoV冠状病毒感染性疾病中的应用。。
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