CN114949194B - 一种用于治疗SARS-CoV-2病毒感染的多肽制剂 - Google Patents
一种用于治疗SARS-CoV-2病毒感染的多肽制剂 Download PDFInfo
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Abstract
本发明公开了一种用于治疗SARS‑CoV‑2病毒感染的多肽制剂，一种用于治疗SARS‑CoV‑2病毒感染的多肽制剂，所述的多肽制剂包含如SEQ ID NO.1‑SEQ ID NO.47所示的多肽。本发明的优点为：安全性好、合成方便。
Description
技术领域
本发明涉及免疫学和生物医药领域，具体地说是一种用于治疗SARS-CoV-2病毒感染的多肽制剂。
背景技术
多肽疫苗是按照病原体抗原基因中已知或预测的某段抗原表位的氨基酸序列，通过化学合成技术制备的肽段混合物。多肽疫苗具有抗病毒、抗肿瘤、抗细菌、抗寄生虫感染功能。由于多肽疫苗价廉、安全、特异性强、容易保存和应用的优点，越来越受到重视。
目前全球研发的SARS-CoV-2疫苗主要包括减毒活疫苗、灭活疫苗、病毒载体疫苗、重组蛋白疫苗、多肽疫苗、DNA疫苗、mRNA疫苗等，上述疫苗均可激活体液免疫产生中和性抗体，但是减毒活疫苗、灭活疫苗、病毒载体疫苗、重组蛋白疫苗、DNA疫苗、mRNA疫苗均无法清除已被病毒感染的细胞。而多肽疫苗可特异性激活T细胞免疫，清除已被病毒感染的细胞，另外，多肽疫苗还具有可快速合成，开发与制备周期较短，可预防、可治疗等优点，因此，本发明提出了一种用于治疗SARS-CoV-2病毒感染的多肽制剂。
发明内容
本发明之目的是弥补上述之不足，向社会公开安全性好、合成方便的一种用于治疗SARS-CoV-2病毒感染的多肽制剂。
本发明的技术方案是这样实现的：
一种用于治疗SARS-CoV-2病毒感染的多肽制剂，所述的多肽制剂包含如SEQ IDNO.1-SEQ ID NO.47所示的多肽，所述的多肽对应为M97-115、M103-121、M166-184、M193-211、N99-117、N160-178、N176-194、N217-235、N235-253、N261-279、N300-318、N311-329、N320-338、N356-374、S52-70、S73-91、S84-102、S128-146、S164-182、S188-206、S264-282、S297-315、S307-325、S356-374、S365-383、S412-430、S484-502、S524-542、S545-563、S630-648、S701-719、S752-770、S777-795、S816-834、S822-840、S864-882、S951-969、S971-989、S978-996、S995-1013、S1015-1033、S1054-1072、S1055-1073、S1089-1107、S1142-1160、S1180-1198以及S1187-1205。
一种用于治疗SARS-CoV-2病毒感染的多肽制剂的应用，应用在药学上可接受的盐或酯或前药，所述的盐或酯或前药包含如SEQ ID NO.1-SEQ ID NO.47所示的多肽。
本发明多肽制剂的开发主要包括以下几个步骤：
步骤一、多肽预测的人工智能算法的优化：
基于人工智能算法和公共数据库中HLA肽段结合数据优化HLA亲和力肽段预测工具。
步骤二、利用人工智能算法预测并筛选对T细胞高亲和力的抗原肽段：
使用SARS-CoV-2病毒的S、E、N、M蛋白序列预测与I类HLA进行亲和力预测，基于肽段与HLA亲和力、肽段的水溶性、免疫原性等指标筛选与HLA结合IC50低于500nM、水溶性强、免疫原性强9氨基酸(9-mer)肽段。最终筛选出47条9氨基酸(9-mer)肽段，在肽段两端延长5个氨基酸，得到对应的19氨基酸(19-mer)肽段用于后续实验。
步骤三、利用化学合成法合成多肽：
利用化学合成法合成预测的SARS-CoV-2特异性多肽，每条肽段包含19个氨基酸，共47条肽段。
步骤四、收集新冠病毒感染康复者的全血，分离PBMC(peripheral bloodmononuclear cells)：
在某医院收集COVID-19康复患者的临床信息与全血样本，全血取于绿色抗凝管中，每个个体收集全血16ml，并于6小时内分离PBMC用于实验或-80℃冻存。
步骤五、HLA分型测序：
提取康复者全血DNA，基于高通量测序技术进行HLA分型鉴定。
步骤六、体外免疫系统激活实验验证HLA分型对应的多肽激活T细胞免疫的有效性：
PBMC细胞与合成的肽段疫苗共培养，采用酶联免疫斑点(Enzyme-linkedimmunospot,ELISpot)方法检测激活的T细胞IFN-γ分泌水平，在体外实验验证多肽的有效性。
本发明与现有技术相比的优点是：
本发明提出一种用于治疗SARS-CoV-2病毒感染的多肽制剂，该多肽是基于人工智能算法预测的SARS-CoV-2特异性抗原肽，具有安全性好，合成速度快的优点。利用多肽刺激HLA-A*02:01、HLA-A*02:07、HLA-A*11:01、HLA-A*11:02、HLA-A*24:02、HLA-A*26:01、HLA-A*30:01、HLA-A*31:01、HLA-A*32:01、HLA-A*33:03分型的SARS-CoV-2感染康复者血液PBMC细胞，均能够检测到T细胞的强烈应答反应。该多肽制剂非常适用目前临床HLA-A*02:01、HLA-A*02:07、HLA-A*03:01、HLA-A*11:01、HLA-A*11:02、HLA-A*24:02、HLA-A*26:01、HLA-A*30:01、HLA-A*31:01、HLA-A*32:01、HLA-A*33:03分型患者的SARS-CoV-2感染的治疗。
附图说明
图1是本发明多肽制剂的研发流程图；
图2是本发明多肽制剂实验结果图；
图3是本发明多肽制剂实验结果统计图。
具体实施方式
下面结合附图进一步详细描述本发明：
一种用于治疗SARS-CoV-2病毒感染的多肽制剂，所述的多肽制剂包含如SEQ IDNO.1-SEQ ID NO.47所示的多肽，所述的多肽对应为M97-115、M103-121、M166-184、M193-211、N99-117、N160-178、N176-194、N217-235、N235-253、N261-279、N300-318、N311-329、N320-338、N356-374、S52-70、S73-91、S84-102、S128-146、S164-182、S188-206、S264-282、S297-315、S307-325、S356-374、S365-383、S412-430、S484-502、S524-542、S545-563、S630-648、S701-719、S752-770、S777-795、S816-834、S822-840、S864-882、S951-969、S971-989、S978-996、S995-1013、S1015-1033、S1054-1072、S1055-1073、S1089-1107、S1142-1160、S1180-1198以及S1187-1205。
一种用于治疗SARS-CoV-2病毒感染的多肽制剂的应用，应用在药学上可接受的盐或酯或前药，所述的盐或酯或前药包含如SEQ ID NO.1-SEQ ID NO.47所示的多肽。
本发明提出一种用于治疗SARS-CoV-2病毒感染的多肽制剂，该多肽是基于人工智能算法预测的SARS-CoV-2特异性抗原肽，具有安全性好，合成速度快的优点。利用多肽刺激HLA-A*02:01、HLA-A*02:07、HLA-A*11:01、HLA-A*11:02、HLA-A*24:02、HLA-A*26:01、HLA-A*30:01、HLA-A*31:01、HLA-A*32:01、HLA-A*33:03分型的SARS-CoV-2感染康复者血液PBMC细胞，均能够检测到T细胞的强烈应答反应。该多肽制剂非常适用目前临床HLA-A*02:01、HLA-A*02:07、HLA-A*03:01、HLA-A*11:01、HLA-A*11:02、HLA-A*24:02、HLA-A*26:01、HLA-A*30:01、HLA-A*31:01、HLA-A*32:01、HLA-A*33:03分型患者的SARS-CoV-2感染的治疗。
本发明基于人工智能算法预测并筛选与HLA高亲和力的SARS-CoV-2病毒特异性抗原肽，在体外进行化学合成后用于刺激人体免疫细胞，激活SARS-CoV-2特异性的细胞免疫。下面通过实验证明本发明多肽制剂在治疗SARS-CoV-2病毒感染上有效性。
如图1所示，本发明多肽制剂的开发过程以下几个步骤：
步骤一、人工智能算法的优化：
构建前馈单层人工神经网络，设定23个参数值作为算法的评分向量。23评分向量输入值均为0.05，另外肽段水溶性、和正常人类蛋白差异等均作为输入层的参数。输入数据集主要使用IEBD数据库和文献提供的质谱数据，并通过已经验证过的数据集验证模型。
步骤二、预测多肽：
通过构建的模型，输入SARS-CoV-2病毒的S、E、N、M蛋白序列和HLA序列信息可以获取与该HLA分型的高亲和力多肽序列，我们挑选排名前0.5％的多肽作为候选肽段。根据多肽与HLA亲和力、肽段的水溶性、免疫原性等指标筛选出与HLA结合IC50低于500nM、水溶性强、免疫原性强的肽段。最终筛选出47条9氨基酸(9-mer)肽段，并得到对应的延长后的19氨基酸(19-mer)肽段。
47条免疫肽段如下：
步骤三、合成多肽：
利用化学合成法合成预测的SARS-CoV-2特异性多肽，分别为M97-115、M103-121、M166-184、M193-211、N99-117、N160-178、N176-194、N217-235、N235-253、N261-279、N300-318、N311-329、N320-338、N356-374、S52-70、S73-91、S84-102、S128-146、S164-182、S188-206、S264-282、S297-315、S307-325、S356-374、S365-383、S412-430、S484-502、S524-542、S545-563、S630-648、S701-719、S752-770、S777-795、S816-834、S822-840、S864-882、S951-969、S971-989、S978-996、S995-1013、S1015-1033、S1054-1072、S1055-1073、S1089-1107、S1142-1160、S1180-1198、S1187-1205，每种肽段纯度＞95％，共合成5mg肽段。取1mg溶于100μl无菌水中，制成母液，接着取一部分母液使用无菌水进行稀释，配制成工作液为200μg/ml。
步骤四、收集新冠病毒感染康复者的全血，分离PBMC(peripheral bloodmononuclear cells)：
在某医院收集COVID-19康复患者血液于绿色抗凝管中，每个个体收集血液16ml，并于6小时内分离PBMC用于实验或-80°冻存。首先将血液转移到干净的50ml离心管中，2000rpm，20℃离心10分钟；收集上层血清，-80℃冻存；向SepMateTM-50管底部加入Ficoll-Paque Premium 1.077溶液，再向沉淀物中加入等体积的PBS，稀释血样并轻轻混合保持试管垂直，将稀释后的样品沿管壁加入SepMateTM-50管中，，1200xg离心15min；然后将上层溶液离心并转移到一个新的50ml无菌离心管中，用PBS清洗沉淀物，共清洗2次；使用红细胞裂解液裂解细胞3分钟；最后用细胞冻存液悬浮细胞，被储存在液氮中，以便将来用于检测或直接用培养基悬浮用于检测。
步骤五、HLA分型鉴定：
使用Ex-DNA全血基因组核酸提取试剂盒和NP968全自动核酸提取仪进行全血DNA的提取。按照说明书要求设置仪器自动化提取程序，DNA提取完成后转移干净无核酸酶离心管中，-20℃保存。利用全血DNA，HiSeq X10等二代测序平台进行高通量测序鉴定HLA分型。本实验涉及的17名康复者HLA分型包括HLA-A*02:01、HLA-A*02:07、HLA-A*11:01、HLA-A*11:02、HLA-A*24:02、HLA-A*26:01、HLA-A*30:01、HLA-A*31:01、HLA-A*32:01、HLA-A*33:03。
步骤六、体外免疫系统激活实验验证HLA分型对应的多肽激活T细胞免疫的有效性：
a)细胞复苏并计数：从液氮中取出冻存PBMC细胞，于37℃水浴快速解冻。完全解冻后，台盼蓝染色计数活细胞数量。
b)细胞培养：按每孔5*10^6个细胞铺于24孔板。加入IL-2(工作浓度20U/ml)、IL-7(工作浓度20ng/ml)、合成的所有肽段(工作浓度2μg/ml)，于37℃(含5％CO2)细胞培养箱培养10天。培养期间每三天半换液。
c)收细胞：培养10天后收取细胞。将细胞转移到离心管中，使用1*PBS清洗细胞3次，以去除培养液中的IFN-γ。使用1640培养液(含10％FBS)重悬细胞，并计数。
d)准备ELISpot板：配置35％乙醇，每孔加入35％乙醇孵育1min。无菌H2O洗涤5遍。每孔加100μl包被抗体(工作浓度15μg/ml)，4-8℃过夜孵育。
e)孵育细胞：用无菌1*PBS洗板5次。每孔加1640培养液(含10％FBS)，置于室温孵育至少30min。弃上清，加入细胞悬浮液，每孔细胞数2×10^4-2.5×10^5。
f)加入刺激物共培养：不含诱导物-H2O(阴性对照组)：加入无菌H2O 2μl；PHA刺激(阳性对照组)：每孔加入10μl PHA(工作浓度2.5ug/ml)；肽段刺激(实验组)：每孔加入47条肽段混合肽池1μl(All pep.Pool：M97-115、M103-121、M166-184、M193-211、N99-117、N160-178、N176-194、N217-235、N235-253、N261-279、N300-318、N311-329、N320-338、N356-374、S52-70、S73-91、S84-102、S128-146、S164-182、S188-206、S264-282、S297-315、S307-325、S356-374、S365-383、S412-430、S484-502、S524-542、S545-563、S630-648、S701-719、S752-770、S777-795、S816-834、S822-840、S864-882、S951-969、S971-989、S978-996、S995-1013、S1015-1033、S1054-1072、S1055-1073、S1089-1107、S1142-1160、S1180-1198、S1187-1205，工作浓度2μg/ml)。把板置于37℃培养箱(5％CO2)，培养12-48小时。
g)显色：吸走细胞，用无菌1*PBS洗板5次。每孔加100μl检测抗体(工作浓度1μg/ml)，室温孵育2h。用无菌1*PBS洗板5次。每孔加100μl Streptavidin-HRP，室温孵育1h。用无菌1*PBS洗板5次。每孔加入显色剂，室温避光孵育5-10min。
h)终止：加入无菌H2O终止。吸走上清，无菌H2O清洗3遍，晾干板，解剖镜下检测并统计斑点数。
i)结果分析：本实验收集17名COVID-19康复者的外周血分离PBMC，利用预测的多肽池进行体外刺激实验。结果发现，HLA分型为HLA-A*02:01、HLA-A*02:07、HLA-A*03:01、HLA-A*11:01、HLA-A*11:02、HLA-A*24:02、HLA-A*26:01、HLA-A*30:01、HLA-A*31:01、HLA-A*32:01、HLA-A*33:03的康复者经多肽池刺激可产生强烈的T细胞反应，且具有统计学意义(如图2和图3所示，图2中Pos.con.为阳性对照结果；All pep.pool为47条肽段共刺激结果；Neg.con.为阴性对照结果；三位野生型新冠病毒感染康复者P01、P02和P03；两位Delta型新冠病毒感染康复者P04和P05)。该肽池可显著引起患者体内的SARS-CoV-2特异性T细胞反应，可用于SARS-CoV-2病毒感染的治疗。
本发明的最佳实施例已被阐明，由本领域普通技术人员做出的各种变化或改型都不会脱离本发明的范围。
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<120> 一种用于治疗SARS-CoV-2病毒感染的多肽制剂
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<212> PRT
<213> Homo sapiens
<400> 47
Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu
1 5 10 15
Leu Gly Lys
Claims (1)
1.一种用于治疗SARS-CoV-2病毒感染的多肽制剂，其特征是：所述的多肽制剂由如SEQID NO.1- SEQ ID NO.47所示的多肽组成，所述的多肽对应为M97-115、M103-121、M166-184、M193-211、N99-117、N160-178、N176-194、N217-235、N235-253、N261-279、N300-318、N311-329、N320-338、N356-374、S52-70、S73-91、S84-102、S128-146、S164-182、S188-206、S264-282、S297-315、S307-325、S356-374、S365-383、S412-430、S484-502、S524-542、S545-563、S630-648、S701-719、S752-770、S777-795、S816-834、S822-840、S864-882、S951-969、S971-989、S978-996、S995-1013、S1015-1033、S1054-1072、S1055-1073、S1089-1107、S1142-1160、S1180-1198以及S1187-1205。
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