CN117430664B - 一种甲型流感病毒t细胞抗原表位肽及其应用 - Google Patents
一种甲型流感病毒t细胞抗原表位肽及其应用 Download PDFInfo
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Abstract
本发明属于免疫治疗技术领域，具体涉及一种甲型流感病毒T细胞抗原表位肽及其应用。本发明要解决的技术问题是目前在甲型流感病毒领域尚没有开发一种用于甲型流感病毒通用疫苗的T细胞抗原表位肽。本发明的技术方案一种甲型流感病毒T细胞抗原表位肽，其氨基酸序列如SEQ ID No.6所示。本发明提供了的抗原表位肽具有很强的免疫原性，能诱导抗原特异性的CD8+T细胞；其可以和HLA‑A2重链和HLA‑A2轻链β2m蛋白组装成pMHC复合物；或直接负荷到抗原递呈细胞，能活化T细胞，有效诱导T细胞免疫，可用于甲型流感病毒通用疫苗研发、制备和药物研发、临床治疗。
Description
技术领域
本发明属于免疫治疗技术领域，具体涉及一种甲型流感病毒T细胞抗原表位肽及其应用。
背景技术
甲型流行性感冒病毒(influenza A virus,IAV，以下简称甲型流感病毒)引起的季节性流感及其并发症依然是人类健康的重大威胁。据世界卫生组织统计，全球每年季节性流感的感染率高达5～15％，约3亿4千万到10亿人口感染流感，有高达25-50万人死于流感，死亡率达到0.1％。虽然大多数地区的流感爆发于冬季，但是对于气候温暖地区，全年任何时刻都可能有流感。因此，流感依然是严重威胁人类健康的，特别是儿童、老人、孕妇和免疫功能低下人群的重要疾病，加强对流感相关的研究依然具有重大的科学和社会意义。
由于有效抗病毒药物的缺失，对流感及其并发症的治疗主要是对症治疗。疫苗接种是目前比较有效的预防措施，多以诱导机体主动产生保护性抗体为主。但是由于甲型流感病毒是单链反义RNA病毒，极易发生变异，产生抗原漂移(antigenic drift)和抗原转变(antigenic shift)，使中和性抗体失去效应，从而导致疫苗失效。相对应的，细胞免疫应答识别高度保守的递呈抗原，因此在不同的甲型流感病毒亚型之间呈现高效的交叉保护能力。譬如，甲型流感病毒(IAV)基质蛋白1(Matrix Protein 1，M1)58到66位GILGFVFTL(M158-66 GIL，简称GIL)特异性的CD8+T细胞免疫应答被发现占据了高达80％的抗甲型流感病毒免疫应答。因此，研究抗甲型流感病毒的细胞免疫应答有望更好地理解抗病毒免疫的机制，为研制广谱高效的抗甲型流感病毒疫苗提供理论与实验基础。
鉴于当前甲型流感病毒变异不断出现，为将来开发通用型甲型流感病毒疫苗和广谱治疗药物提供了契机。通用疫苗是一类选择即使病毒抗原发生变异后仍能有效保护免疫人群特性的广谱疫苗。因此，开发出在甲型流感病毒发生各类变异后，依旧能够给疫苗免疫人群提供有效免疫保护的通用疫苗以及广谱治疗性药物迫在眉睫。
研究显示，T细胞免疫应答在病毒感染后机体抗病毒防御以及机体免疫病理损伤过程中发挥了重要的作用，尤其是CD8+T细胞，其抗原特异性免疫活性11年后依然存在，说明了CD8+T细胞免疫应答在抗甲型流感病毒免疫防御中的重要作用及其在疫苗研发中的重要地位。CD8+T细胞免疫应答的第一步就是T细胞通过其表面的抗原识别受体特异性识别被病毒感染的细胞所递呈的抗原表位肽。因此，抗原表位肽是T细胞特异性识别病毒、发挥免疫保护作用的重要关键分子，是免疫检测、免疫治疗和疫苗研发的关键靶向分子。
CD8+T细胞通过T细胞受体(TCR)识别pMHC活化，杀灭病毒感染细胞、清除病毒，从而发挥抗病毒的细胞免疫作用。因此，鉴定可以有效活化T细胞抗原表位肽，在甲型流感病毒病毒抗原发生变异后仍能诱导有效T细胞免疫保护，是开发甲型流感病毒通用型疫苗和广谱治疗性药物的关键之一。
免疫逃逸是免疫抑制病原体通过其结构和非结构产物，拮抗、阻断和抑制机体的免疫应答。甲型流感病毒因为经常地持续性地发生突变，产生了大量的变异株，严重的威胁了疫苗的免疫屏障效果。
发明内容
本发明要解决的技术问题是目前在甲型流感病毒领域尚没有开发一种用于甲型流感病毒通用疫苗的T细胞抗原表位肽。
本发明的技术方案一种甲型流感病毒T细胞抗原表位肽，其氨基酸序列如SEQ IDNo.6所示。
进一步的，本发明还提供了编码所述抗原表位肽的核酸分子。
本发明还提供了含有所述抗原表位肽的pMHC复合物。
进一步的，所述pMHC复合物由HLA-A2重链、HLA-A2轻链β2m和所述抗原表位肽复性得到。
其中，HLA-A2重链、HLA-A2轻链β2m和所述抗原表位肽的摩尔比为1︰2︰10。
本发明还进一步提供了一种抗原肽-抗原递呈细胞复合物，为表面负荷所述抗原表位肽的抗原递呈细胞。
其中，所述抗原递呈细胞为CD8+T细胞。
优选的，所述CD8+T细胞为T2-A2细胞。
本发明还提供了上述抗原表位肽、编码所述抗原表位肽的核酸分子、pMHC复合物和/或抗原肽-抗原递呈细胞复合物在制备甲型流感病毒药物中的应用。
本发明还提供了上述抗原表位肽、编码所述抗原表位肽的核酸分子、pMHC复合物和/或抗原肽-抗原递呈细胞复合物在筛选甲型流感病毒药物中的应用。
本发明还进一步提供了上述抗原表位肽、编码所述抗原表位肽的核酸分子、pMHC复合物和/或抗原肽-抗原递呈细胞复合物在制备甲型流感病毒疫苗中的应用。
本发明还进一步提供了上述抗原表位肽、编码所述抗原表位肽的核酸分子、pMHC复合物和/或抗原肽-抗原递呈细胞复合物在制备评估甲型流感病毒疫苗接种效果的药物的应用。
本发明的有益效果：本发明提供了一种甲型流感病毒T细胞抗原表位肽，其具有很强的免疫原性，能诱导抗原特异性的CD8+T细胞；其可以和HLA-A2重链和HLA-A2轻链β2m蛋白组装成pMHC复合物；或直接负荷到抗原递呈细胞，能活化T细胞，有效诱导T细胞免疫，避免甲型流感病毒突变株的免疫逃逸，并在流感疫苗接种者和康复者中均可检测到特异性的CD8+T细胞，可用于甲型流感病毒通用疫苗研发、制备和药物研发、临床治疗。
本发明还利用该甲型流感病毒CD8+T细胞抗原表位肽制备成带有PE荧光通道的pMHC复合物，可以用于甲型流感病毒疫苗接种者和感染康复者外周血内抗原特异性T细胞的检测，并用于体外的T细胞活化实验，这些甲型流感病毒CD8+T细胞抗原表位肽可以用于制备针对各种甲型流感病毒突变株的通用疫苗、甲型流感病毒相关的免疫检测以及广谱治疗性药物。这些甲型流感病毒CD8+T细胞抗原表位肽制备成带有PE荧光通道的pMHC复合物，或直接负荷到抗原递呈细胞，从而活化T细胞，可用于甲型流感病毒疫苗研发、制备和药物研发、临床治疗，可应用于：
1)甲型流感病毒疫苗的研发和制备：甲型流感病毒发生变异后，该多个T细胞表位能诱导机体产生免疫应答生成抗原特异性T细胞。故该T细胞表位是甲型流感病毒通用疫苗的候选抗原表位肽。
2)检测是否具有抵抗甲型流感病毒感染的细胞免疫功能：待检者体内检测到甲型流感病毒抗原特异性T细胞，代表机体已经产生了T细胞免疫功能，根据该抗原表位肽制备成荧光通道的pMHC复合物标记的抗原特异性CD8+T的比例，可以评价机体T细胞免疫功能强弱及对甲型流感病毒感染可能性。
3)评估疫苗接种后的效果：接种者体内检测到甲型流感病毒抗原特异性T细胞，代表机体已经产生了T细胞免疫功能，根据其比例，可以评估机体再次感染甲型流感病毒可能性。
4)监测病情：可用于对密切接触者、医学观察者、疑似和确诊患者的病情变化监测。
5)预后判断：如果机体不能产生T细胞免疫应答，或抗原特异性T细胞比例持续减少，则预示预后不佳。
附图说明
图1、甲型流行性感冒病毒(IAV)T细胞抗原表位的筛选和鉴定，A和B为T2稳定性实验。A为抗β2m的流式细胞检测，B为A的统计图。C：光敏肽置换实验ELISA的结果。Blank(空白对照):no peptides；Negative ctrl(阴性对照)：EBV virus peptide IVTDFSVIK；Positive ctrl(阳性对照)，influenza A M1 peptide GILGFVFTL(下同)。
图2、HLA-A2的pMHC复合物的制备。A为pMHC复合物分子筛(Sephacryl S-200)纯化结果四份不同样品(图中数字标识为1，2，3，4)，数字1-4代表采集的第一到第四个峰值。B为考马斯蓝染色的15％ SDS-PAGE结果；M：蛋白分子量marker，图中数字1-4对应图A中1-4的峰，其中红色标记的2代表目的峰，为HLA的重链(a)和轻链(β2m)。
图3、甲型流感病毒T细胞抗原表位的免疫原性鉴定。A和B：16小时后CD69(上排)和CD137(下排)分子的检测，B为A的统计图。
图4、抗原表位混合刺激后检测到抗原特异性T细胞的生成；A为抗原表位混合刺激健康人CD8+T细胞0天(上排)和7天(下排)后带有荧光通道的pMHC复合物可以检测到抗原特异性T细胞的生成，B为A的统计图。
图5、表位刺激的T细胞介导的T2细胞凋亡比例。A-B(上排)：刺激培养7天时间T2A2细胞的凋亡百分比；(下排)第7天末，T2A2细胞凋亡百分比。
图6、检测活化CD8+T细胞中IFN-γ(上排)和Granzyme B(下排)的表达情况(A)。B为A的统计图。
图7、评估流感灭活疫苗接种后14天和甲型流感病毒感染康复者7天后体内特异性CD8+T细胞的比例；A-B：流式细胞术检测HLA-A2+流感灭活疫苗接种者上述4种(T细胞活化时，可以产生的抗原特异性CD8+T表位)肽的特异性CD8+T细胞；C-D：流式细胞术检测HLA-A2+甲型流感病毒感染康复者上述4种(T细胞活化时，可以产生的抗原特异性CD8+T表位)肽的特异性CD8+T细胞。
具体实施方式
T2-A2细胞是一种通过重组基因工程技术表达人MHC-I类分子HLA-A2的抗原递呈细胞系。只有有效的抗原表位肽才能被其递呈，从而在其细胞表面形成稳定的pMHC复合物，因此可以作为刺激T细胞的人工抗原递呈细胞。
T细胞抗原表位肽单独无法工作，必须以pMHC复合物或抗原肽-抗原递呈细胞复合物的方式进行T细胞活化。本发明利用MHC的单体和鉴定出的甲型流感病毒T细胞表位进行联合复性，制备了pMHC复合物。将鉴定出的甲型流感病毒T细胞抗原表位肽负荷在抗原递呈细胞表面(T2-A2细胞)，制备了抗原肽-抗原递呈细胞复合物，然后用制备好的pMHC复合物去标记CD8+T细胞，发现其抗原表位肽可以有效活化健康人外周血中T细胞，并具有很强的免疫原性，也可以有效杀伤携带甲型流感病毒抗原的靶细胞。该T细胞抗原表位肽组装成的带有PE荧光通道的pMHC复合物，可以在甲型流感病毒疫苗接种者和康复者中均可检测到。证明新发现的甲型流感病毒CD8+T细胞抗原表位肽能有效诱导T细胞免疫，避免病毒突变的免疫逃逸，可以作为通用型疫苗或应用于免疫治疗等。
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1甲型流感病毒HLA-A2限制性抗原表位肽的预测
以美国NIH抗原表位数据库(IEDB)提供的MHC-I类分子预测工具(http://tools.iedb.org/mhci/)进行HLA-A2限制性抗原表位预测。招募的志愿者是接种甲型流感病毒裂解疫苗，疫苗采用毒诸位采用WHO和欧盟推荐的A1型、A3型和B型甲型流感病毒(2021/2022)的流行株或相似株(北半球)。疫苗的主要成分是含有以下毒株(2021/2022)的抗原(an A/Victoria/2570/2019(H1N1)pdm09；an A/Cambodia/e0826360/2020(H3N2)；aB/Washington/02/2019(B/Victoria lineage)-like virus)。选用最常见的H1N1和H3N2在Gisaid数据库中找到对应的序列编号(EPI_ISL_417210；EPI_ISL_806547)进行抗原表位蛋白的预测。最后得到11种候选的甲型流感病毒特异性HLA-A2+的CD8+T细胞抗原表位肽，如表1所示。
表1甲型流感病毒病毒T细胞11种抗原表位肽
| 编号 | 开始位置 | 结束位置 | 长度 | 序列 | 序列号 |
| F1 | 413 | 421 | 9 | NMLSTVLGV | SEQ ID No.1 |
| F2 | 46 | 54 | 9 | FMYSDFHFI | SEQ ID No.2 |
| F3 | 18 | 26 | 9 | STICFFMQI | SEQ ID No.3 |
| F4 | 275 | 283 | 9 | CLPACVYGL | SEQ ID No.4 |
| F5 | 373 | 381 | 9 | AMDSNTLEL | SEQ ID No.5 |
| F6 | 24 | 32 | 9 | MQIAILITT | SEQ ID No.6 |
| F7 | 458 | 466 | 9 | FQGRGVFEL | SEQ ID No.7 |
| F8 | 122 | 130 | 9 | AIMDKNIIL | SEQ ID No.8 |
| F9 | 218 | 226 | 9 | CVNGSCFTV | SEQ ID No.9 |
| F10 | 249 | 257 | 9 | KADTKILFI | SEQ ID No.10 |
| F11 | 128 | 136 | 9 | IVLEANFSV | SEQ ID No.11 |
实施例2甲型流感病毒HLA-A2限制性抗原表位肽的鉴定
人工合成实施例1预测得到的候选T细胞抗原表位肽(南京金斯瑞生物科技有限公司)，配置成浓度为20μM。取对数生长状态T2-A2细胞(T2-A2为马萨诸塞大学医学院(University of Massachusetts Medical School)Anna Gil博士的赠与)，种植到96孔板，每孔105个，分布设置空白孔、阴性对照肽(EB病毒，IVTDFSVIK)、阳性对照肽(甲型流感M1肽，SEQ ID No.12：GILGFVFTL)和各合成候选T细胞抗原表位肽，每组3个复孔，终体积200μL。37℃孵育4小时后离心洗涤两次，以FITC anti-human HLA-A2(β2m)抗体标记，4℃避光孵育30分钟后，以流式细胞仪检测。实验共进行3次。
结果如图1的A和B所示，结果显示，11种抗原多肽均可以被抗原递呈细胞有效递呈信息给T细胞，说明这些肽是T细胞抗原表位肽。
实施例3抗原多肽形成pMHC复合物的检测
采用ELISA法检测实施例2中筛选得到的11种甲型流感病毒T细胞抗原表位肽。具体操作如下：
在室温下，用100μL的0.5μg mL-1链霉亲和素在96个U形板上孵育16～18小时，用洗涤缓冲液(BioLegend,Cat#420201,US)洗涤3次，并在室温下用稀释缓冲液(0.5M Tris pH8.0，1M NaCl，1％ BSA，0.2％吐温20)封闭30分钟。光敏肽(SEQ ID No.13：KILGFVFJV)与MHC((BioLegend,Cat#280003,US))形成的pMHC复合物设为HLA空白对照，甲型流感M1肽(GILGFVFTL)设为阳性对照，EB病毒肽(SEQ ID No.14：IVTDFSVIK)设为阴性对照。然后，分别添加用365nm三用紫外线分析仪(Qilin Bell，Cat#1903274)置换好的20μL实施例1中的11种流感病毒T细胞抗原表位肽的稀释液(400μM)和20μL Flex-TTM单体(200μg/mL)的稀释液100μL到96孔板，并在温度为37℃的细胞培养箱里孵育1小时。使用洗涤缓冲液洗涤3次后，添加100μL稀释HRP结合抗体(BioLegend,Cat#280303,US)，并在37℃下继续孵育1小时，然后洗涤。添加100μL底物溶液(10.34mL去离子水，1.2mL 0.1M柠檬酸一水合物/柠檬酸三钠二水合物，pH 4.0，240μL 40mM ABTS，120μL过氧化氢溶液)，并在室温避光孵育8min。使用50μL终止溶液(2％w/v草酸二水合物)终止反应。在30分钟内，用酶标仪在450nm波长下测定吸光度(OD值)。
结果如图1的C所示，结果显示11种抗原多肽均可以形成pMHC复合物。
实施例4抗原表位肽的pMHC复合物单体的制备
将HLA-A2α链和β2m的核苷酸序列分别构建到pET28a表达载体上，转入大肠杆菌BL21(DE3)进行蛋白的表达，使用镍柱(Biorad，Cat#7324610，US)亲和层析对HLA-A2蛋白(HLA-A2重链a链、HLA-A2轻链β2m)进行纯化，将纯化的蛋白HLA-A2α链蛋白、β2m蛋白和实施例1中表1中11种抗原表位肽分别按照1︰2︰10的摩尔比逐步滴加至40mL复性溶液(5M尿素、0.4M精氨酸、100mM Tris、3.7mM胱胺、6.3mM半胱胺和2mM EDTA)中进行复性，得到抗原表位肽的pMHC复合物单体。
pMHC复合物单体经过DEAE离子交换柱进一步纯化，以0.5M NaCl洗脱，并根据OD280nm紫外吸收峰收集蛋白。随后经DEAE离子交换柱纯化后的蛋白根据分子量大小，通过Superdex 75pg分子筛纯化，以PBS洗脱，并根据OD280 nm紫外吸收峰收集不同分子量蛋白。图2A和2B所示，经过纯化后得到了抗原表位肽的pMHC复合物三聚体(HLA-A2重链a链+HLA-A2轻链β2m+抗原肽的三聚体)。
实施例5甲型流感病毒HLA-A2限制性抗原表位肽活化T细胞
T2细胞通过表达HLA-A2分子(T2-A2，PMID:34414379；PMID:35194575；PMID:35116022；PMID:37117789)来活化T细胞。分离健康志愿者的外周静脉血中的单个核淋巴细胞(PBMCs)，并进一步分离CD8+T细胞。将T2-A2细胞用CFSE标记，用20μg/mL丝裂霉素处理20分钟后，分别用实施例1中11种不同抗原肽孵育。
具体做法为：
将96孔板中每个孔种0.5×106个CD8+T细胞分别与表1中的11条抗原表位肽负荷的0.5×106个T2-A2细胞共培养(共11个孔)，并用1μg/mL抗人CD28抗体和50IU/mL IL-2共刺激。每隔两天补充50IU/mL IL-2和20μM抗原表位肽。
分离健康志愿者外周静脉血中CD8+T细胞，和抗原多肽负荷的T2细胞共同培养，加入1μg/mL抗人CD28抗体和50IU/mL IL-2共刺激，培养16小时后检测T细胞活化分子CD69和CD137的表达。
将实施例4得到的30μL抗原表位肽的pMHC复合物单体与3.3μL PE链霉亲和素(BioLegend Cat#405203，US)在96孔板中混合，并在4℃避光孵育30分钟后，再加入2.4μL封闭溶液(1.6μL 50mM生物素(Thermo Fisher，Cat#B20656，US))和198.4μL PBS以停止反应，并在4～8℃下培养过夜，即可得到带有PE荧光通道的pMHC复合物。
培养7天后计算T2-A2的存活数目百分比、用带有PE荧光通道的pMHC复合物标记特异性CD8+T细胞比例和T2-A2细胞上的凋亡标记物Annexin V-APC百分比，检测特异性CD8+T细胞释放IFN-γ和GZMB的情况。与CD8+T细胞培养7天后IAV介导T2凋亡百分比：CFSE标记的T2A2作为靶细胞，检测残留的靶细胞数目，计算方法为T2细胞的50％减去存活细胞的百分比。
结果如图3A和3B所示，实施例2中筛选得到的11种甲型流感病毒T细胞抗原表位肽均能够活化T细胞。其中，F3对应的T细胞抗原表位肽STICFFMQI、F6对应的T细胞抗原表位肽MQIAILITT、F10对应的T细胞抗原表位肽KADTKILFI和F11对应的T细胞抗原表位肽IVLEANFSV活化CD8+T细胞的能力较强，如图4A和4B所示。同时以上11条肽活化的特异性CD8+T可以杀伤靶细胞，如图5A和5B所示；特异性的CD8+T细胞释放IFN-γ和GZMB，如图6A和6B所示。
实施例6甲型流感病毒HLA-A2限制性抗原表位肽活化T细胞
分离甲型流感病毒接种灭活疫苗第二针后14天和甲型流感病毒感染康复者7天后的志愿者外周静脉血中的PBMCs，并鉴定其HLA亚型，HLA-A2阳性PBMCs样品进一步用实施例5中带有PE荧光通道的pMHC复合物和CD8-APC抗体进行染色，然后进行流式上机检测。
结果如图7所示，结果显示，F3、F6、F10和F114种抗原表位肽的带有荧光通道的pMHC复合物可以识别甲型流感病毒疫苗接种者(图7A和7B)和甲型流感病毒感染康复者(图7C和7D)体内产生的抗原特异性CD8+T细胞。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
Claims (7)
1.一种甲型流感病毒T细胞抗原表位肽，其特征在于：其氨基酸序列如SEQ ID No.6所示。
2.编码权利要求1所述抗原表位肽的核酸分子。
3.含有所述权利要求1所述抗原表位肽的pMHC复合物，其特征在于：由HLA-A2重链、HLA-A2轻链β2m和所述抗原表位肽复性得到。
4.根据权利要求3所述pMHC复合物，其特征在于：HLA-A2重链、HLA-A2轻链β2m和所述抗原表位肽的摩尔比为1︰2︰10。
5.一种抗原肽-抗原递呈细胞复合物，其特征在于：为表面负荷权利要求1所述抗原表位肽的抗原递呈细胞；所述抗原递呈细胞为T2-A2细胞。
6.权利要求1所述抗原表位肽、权利要求2所述编码抗原表位肽的核酸分子、权利要求3或4所述pMHC复合物和/或权利要求5所述抗原肽-抗原递呈细胞复合物在制备甲型流感病毒药物中的应用。
7.权利要求1所述抗原表位肽、权利要求2所述编码抗原表位肽的核酸分子、权利要求3或4所述pMHC复合物和/或权利要求5所述抗原肽-抗原递呈细胞复合物在制备甲型流感病毒疫苗或制备评估甲型流感病毒疫苗接种效果的药物中的应用。
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