CN113181355A - 一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗及其制备方法 - Google Patents
一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗及其制备方法 Download PDFInfo
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Abstract
本发明属于生物医药领域,具体涉及一种DC细胞靶向的纳米SARS‑CoV2S蛋白多肽池疫苗及其制备方法,本发明通过甘露糖修饰纳米颗粒,使疫苗特异性靶向DC细胞,加速DC细胞对SARS‑CoV2抗原的获取,促进DC细胞成熟,高效递呈抗原给CTL细胞,促进CTL细胞增殖,增强CTL细胞杀伤作用,从而提高多肽疫苗的免疫效力。本发明利用生物信息学技术进行多肽预测分析,利用纳米颗粒靶向技术高效激活DC细胞,有效激活抗SARS‑CoV2细胞免疫作用,为防控COVID‑19提供更加安全有效的DC细胞靶向的纳米SARS‑CoV2S蛋白多肽池疫苗。
Description
技术领域
本发明属于生物医药领域,具体涉及一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗及其制备方法。
背景技术
目前中国并行推进灭活疫苗、腺病毒载体疫苗、重组蛋白疫苗、核酸疫苗、减毒流感疫苗等五条技术路线研发,已有3种疫苗获得有条件使用。灭活疫苗指选用免疫原性强的病原体,经人工大量培养后,用物理或化学方法将其灭活,破坏病毒的复制能力,使其失去致病性但保留免疫原性。灭活疫苗具有技术路线成熟、早期研发速度快、不能在体内繁殖,接种后不会导致相应的疾病等优点;灭活疫苗质控点和评价方法也比较明确,具有较好的安全性,但灭活疫苗往往需要进行毒株的分离培养,对实验室的生物安全级别要求较高,难以实现生产阶段的产能迅速放大,且免疫效力较低,需多剂次接种;腺病毒载体疫苗易于生产制备,无需佐剂,安全性相对较高,可诱导产生细胞免疫和粘膜免疫,但人群中普遍存在针对腺病毒的中和抗体,可能会削弱相应的免疫应答,从而影响疫苗的保护效果。
基因工程重组亚单位疫苗是用基因工程方法或分子克隆技术,将病毒的保护性抗原基因构建在表达载体上,再转化至真核或原核细胞中表达出抗原蛋白,最后纯化而成。相比于灭活疫苗,通过合成产生的基因工程亚单位疫苗更具有安全性,因为它不含病毒基因组、不涉及细胞衍生材料、纯度可以控制。但亚单位疫苗免疫效果较差,往往需要多剂次接种或添加免疫佐剂以增强其免疫原性;核酸疫苗又称为基因疫苗,是将含有编码某种抗原蛋白的质粒DNA或mRNA的重组质粒载体通过肌肉注射等方式导入宿主细胞,并在宿主细胞内表达抗原蛋白,从而诱导宿主产生相应的免疫应答。与传统疫苗相比,基因疫苗生产成本更低、更容易纯化、可产生同种异株交叉保护、免疫保护力强。针对此次疫情的突发紧急情况,众多的疫苗研发科研团队选择了此疫苗技术路线。但核酸疫苗免疫原性较弱,不易产生黏膜免疫反应,且核酸疫苗的安全性也有待考究。
DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗以其安全、有效和质量可控的优势可能成为防控COVID-19的候选疫苗。面对COVID-19,目前尚未研制出特效的治疗药物,关键是研究安全有效的疫苗才能阻止疫病的传播。
发明内容
研究抗SARS-CoV2感染多肽疫苗的关键是从SARS-CoV2感染冠状病毒的功能蛋白序列中找出有免疫原性的抗原表位或抗原决定簇。运用生物信息学技术对抗原蛋白的抗原表位进行预测,是确定抗原表位最有效的方法。S蛋白(Spike protein)是位于 SARS-CoV2表面的一种重要的结构蛋白,在病毒与宿主细胞表面受体结合及介导膜融合并进入细胞的过程中起关键性作用,包含病毒与宿主细胞膜受体的结合位点和主要的中和抗原,是进行抗SARS-CoV2感染疫苗设计的重要位点。
为了克服现有技术存在的不足,本发明运用生物信息学技术对S蛋白进行HLA-A2限制性CTL细胞表位预测分析与设计,并合成设计的候选抗原表位多肽。本发明包括纳米颗粒的制备并进行甘露糖修饰,COVID-19多肽表位的选择及纳米颗粒包裹多肽的条件优化,检测纳米多肽疫苗对DC的活化,活化后的DC对T细胞的激活作用及 T细胞对SARS-CoV2多肽冲击的T2靶细胞的杀伤,获得DC细胞靶向的纳米 SARS-CoV2 S蛋白多肽疫苗。
本发明的上述目的是通过以下技术方案实现的:
本发明提供一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,具体包括以下步骤:
步骤S1:称取明胶于烧杯中,加水搅拌溶解,边搅拌边加入丙酮,室温静置,弃去上清液;再加水加温彻底溶解底部沉淀,加入HCl调节pH,再次滴加丙酮,加入用丙酮稀释的戊二醛,室温持续搅拌,挥发丙酮及戊二醛,用双蒸水稀释,使用透析袋,在双蒸水中透析后,在-4℃静置保存;
步骤S2:取步骤S1制备的明胶纳米颗粒于烧杯中,加入甘露糖,NaAc溶液溶解,调节pH,静置一段时间,再持续搅拌2天,透析袋透析24h,4℃保存;
步骤S3:获取SARS-CoV2的S蛋白的氨基酸序列,利用人工神经网络进行S蛋白针对人HLA-A2的MHC-Ⅰ分子结合力预测,筛选亲和力高的表位,并对筛选的表位进行过敏原筛选及理化性质分析。通过对多肽表位进行打分,将多肽表位进行过敏原检测,去除可能成为过敏原的多肽表位,得到序列后制成多肽;另外设计一组MHC- Ⅱ分子限制性Th细胞表位,为15个氨基酸的多肽表位;
步骤S4:调节明胶纳米颗粒溶液pH,向纳米颗粒中分别加入多肽,4℃震荡4h。
进一步的,步骤S1中明胶取0.5g,首次加入10mL双蒸水溶解,再次加入10mL 双蒸水50℃彻底溶解。
进一步的,步骤S1中两次丙酮的用量分别为5~30mL,步骤S1中加入1mol/L 的HCl调节pH至2.5。
进一步的,步骤S1中加入用2mL丙酮稀释的0.5%的戊二醛。
进一步的,步骤S1与步骤S2中使用40KD的透析袋。
进一步的,步骤S2中取1mg/mL明胶纳米颗粒,0.68g甘露糖使用2mLNaAc 溶解。
进一步的,步骤S3中分别利用IEDB网站和NetMHC 4.0 Server网站预测I类MHC 分子结合位点,和对亲和力进行筛选。
进一步的,步骤S3中采用SYFPEITHI方法进行打分。
进一步的,步骤S4中纳米颗粒浓度为1mg/mL,多肽浓度为10g/mL。
本发明提供一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗,如所述的步骤S1-S4的制备方法制备。
本发明与现有技术相比的有益效果是:
(1)与常规的灭活病原体、亚单位及重组疫苗相比,多肽疫苗的优势在于:诱导对完整的抗原分子中表现出弱免疫原性的结构的免疫应答;生产技术安全,可靠;高度标准化;去除具有高副反应的成分,如脂多糖、毒素等;去除对免疫个体可能具有的致敏性成分;将不同抗原得到的各种多肽可以包裹在一种载体中;能够针对复杂的非连续性天然抗原决定簇,构建相应的合成抗原多肽。
(2)纳米靶向DC细胞多肽疫苗启动、活化DC细胞,进而激活免疫系统,能提高DC细胞对抗原的摄取和递呈效率,增强疫苗的免疫效应。纳米颗粒经甘露糖修饰可以发挥佐剂的作用,通过多种途径提高COVID-19疫苗的免疫效应和治疗效果,具体效果如下:
①提高疫苗DC细胞靶向性,加快DC对抗原的获取及呈递,经过甘露糖修饰的纳米颗粒在DC细胞中获取时间由24h缩短至6h,速率是原来的4倍;
②利用纳米颗粒靶向技术高效激活DC细胞,替代传统的利用细胞因子,如TNFα,IFNγ,进行激活的手段,有效激活抗SARS-CoV2细胞免疫作用,为防控COVID-19 提供更加安全有效的多肽池疫苗。在本发明中,经甘露糖修饰的纳米多肽池疫苗对DC 细胞激活48h后,DC细胞表型CD1a、CD11c、CD80、CD83和HLA-DR的表达均大幅度提高,是直接用多肽刺激DC细胞的2倍;
③经过纳米颗粒激活后的DC细胞,对T细胞的增殖和激活作用明显增强,T细胞增殖量提高1.5倍,激活后的T细胞对靶细胞的杀伤率提高77%。
(3)本发明加速启动免疫应答,及时清除SARS-CoV2病原,为COVID-19防控提供了新的解决方案。由于SARS-CoV2感染上皮细胞和免疫细胞,使免疫细胞减少,如单核细胞、巨噬细胞和DC细胞,使这些免疫细胞不能启动免疫应答。尤其是DC 细胞,是启动免疫应答、调节和发挥抗SARS-CoV2细胞免疫的重要细胞。通常情况下,对于COVID-19免疫应答,其始动环节如果DC细胞没有充分激活,造成免疫低下,是机体不能及时清除病原造成疫病持续性感染的主要原因。
附图说明
图1为MnGNP纳米颗粒粒径和Zeta电位;
图2为甘露糖修饰明胶纳米颗粒扫描电镜图;
图3为红外光谱图;
图4为多肽包封率;
图5为多肽释放率;
图6为MoDC细胞激活细胞因子ELISPOT结果;
图7为MoDC细胞激活细胞因子ELISPOT示意图;
图8为纳米疫苗诱导MoDC激活和分化;
图9为纳米疫苗刺激DC细胞成熟12h 400X形态变化;
图10为T细胞增殖刺激指数;
图11为CTL细胞杀伤百分率;
图12为纳米靶向的COVID-19多肽疫苗工作原理示意图。
具体实施方式
下面通过具体实施例详述本发明,但不限制本发明的保护范围。如无特殊说明,本发明所采用的实验方法均为常规方法,所用实验器材、材料、试剂等均可从商业途径获得。
实施例1明胶纳米颗粒的制备,甘露糖修饰明胶纳米颗粒,纳米颗粒包裹多肽
称取0.5g明胶于100mL烧杯中,加入10mL双蒸水,于50℃搅拌溶解,边搅拌边加入10mL丙酮,室温静置20min,弃去上清液,再加入10mL双蒸水,50℃彻底溶解底部沉淀,加入1mol/L的HCl溶液,调节pH至2.5;滴加丙酮30mL,再加入 2mL用丙酮稀释的含0.5%的戊二醛,室温1000rpm持续搅拌5h,挥发丙酮及戊二醛后用双蒸水稀释10倍,使用40KD透析袋,在双蒸水中透析24h后制得明胶纳米颗粒,在-4℃静置保存。
取20mL浓度为1mg/mL明胶纳米颗粒于50mL烧杯中,加入0.68g甘露糖,加入2mLpH4.0、浓度为1mol/L的NaAc溶解甘露糖,37℃静置30min,500rpm持续搅拌2天,40kD透析袋透析24h,4℃保存,利用激光粒度仪进行检测粒径大小及Zeta 电位。
图1为纳米颗粒检测结果,制备的纳米颗粒形态规整,粒子大小均一,带电荷数最大。其粒径为337.25±3.3nm,Zeta电位为-24.36±0.4mV,PDI为0.044±0.028。
将甘露糖修饰后的纳米颗粒利用真空冷冻干燥仪进行冷冻干燥,使用压片法红外光谱检测甘露糖修饰情况,以及扫描电镜检测纳米颗粒的形貌,得到图2与图3。
图2为红外光谱检测甘露糖修饰情况,通过对普通明胶纳米颗粒和甘露糖修饰明胶纳米颗粒的红外光谱研究,证实了甘露糖与明胶纳米颗粒的耦合作用。明胶纳米颗粒的红外光谱在3250-3450cm-1处表现出较弱的N-H拉伸,在1655cm-1处表现出强的 N-H弯曲,表明存在伯胺基。用pH4.0的醋酸钠缓冲液对甘露糖醛基团进行开环及后续反应,制成甘露糖包衣。仲胺在1543cm-1处的N-H弯曲和在1450cm-1处的C=N 拉伸揭示了席夫碱的形成,即形成了RCH=NR键,证实了甘露糖配体与GNP的胺端部的连接形成。此外,在3200-3600cm-1与1083cm-1处的甘露糖的O-H宽强拉伸和 C-O强拉伸也证明了在MnGNP中存在甘露糖中的羟基。
调节明胶纳米颗粒溶液pH7.0,向浓度为1mg/mL的纳米颗粒中加入终浓度为10 μg/mL的多肽,室温震荡2h。
实施例2多肽表位的预测、分析、筛选及合成
研究多肽表位疫苗的关键就是从COVID-19冠状病毒的功能蛋白序列中找出有免疫原性的抗原表位,运用生物信息学技术对S蛋白进行抗原表位预测。
通过NCBI查找病毒相应蛋白氨基酸序列后,对HLA-A2限制性CTL细胞表位进行预测,利用IEDB网站和NetMHC 4.0Server网站预测I类MHC分子结合位点。对获得表位进行交叉筛选亲和力高的表位,%Rank<0.5的表位易形成强键,0.5<%Rank <4易形成弱键;IC 50值<50nM的肽视为高亲和力,<500nM为中间亲和力,<5000 nM低亲和力;接着对筛选的表位进行过敏原筛选及理化性质分析,最后通过 SYFPEITHI方法对多肽表位进行打分,多肽表位得分≥20时被认为与MHC有高结合力,从而获得最佳多肽序列,见表2。
表2表位筛选结果
选取最佳多肽序列进行合成,分别为PEP1,PEP2,PEP3,PEP4;另外,利用IEDB 网站进行MHC-Ⅱ分子限制性表位预测,选取得分最高的表位进行合成,多肽序列为 PEP5;将一个CTL表位与Th表位用柔性连接子AAA连接,多肽序列为PEP6,见表 3。
表3合成肽序列
实施例3DC靶向纳米颗粒的包封率及释放速率检测包封率
将1mg/mL的MnGNP与10μg/mL的荧光标记多肽混合,4℃条件下在微量振荡器上振荡包裹后,10000rpm离心10min,检测上清液及沉淀荧光强度,计算纳米颗粒的包封率。结果如图4。
包封率=沉淀中多肽/加入多肽总量×100%。结果表明多肽的最大包封率为49%。
释放速率:将包裹多肽的明胶纳米颗粒放入37℃恒温摇床中,在0h,3h,6h,9h,12h,24h时依次取出,10000rpm,离心10min,分别检测沉淀及上清液的荧光强度,每次检测后沉淀用等量PBS重悬后重新放入37℃恒温摇床中,待下次继续检测。设置不同时间段对沉淀中纳米颗粒包裹的多肽释放量进行检测,计算其在不同时间多肽的释放率,释放率=上清中多肽含量/多肽总量×100%,其释放情况如图5。
实施例4DC靶向纳米多肽疫苗激活MoDC细胞对T细胞产生IFNγ的影响
将DC靶向纳米多肽疫苗激活MoDC细胞2天后,与T细胞共培养,利用ELISPOT 检测试剂盒对IFNγ含量进行检测。结果表明,相比于其他组,纳米颗粒包裹SARS-CoV2 S蛋白多肽池组激活MoDC细胞,刺激T细胞分泌IFNγ明显升高,表明DC靶向纳米多肽疫苗激活MoDC细胞,刺激T细胞作用增强,ELISPOT结果见图6与图7。
实施例5DC靶向纳米多肽疫苗刺激MoDC细胞成熟,引起表面标志物改变
经过DC靶向纳米多肽疫苗刺激2天后,利用流式检测CD1a、CD11c、CD80、 CD83和HLA-DR的表达情况,见图8。
目前鉴定人DC细胞的主要特征性标志为CD1a、CD80、CD86、CD83、CD11c、 MHCⅡ等分子,其中CD1a是鉴定人外周血与骨髓DC最好的标记,用于DC细胞计数;CD83为DC细胞成熟的标志;CD80和CD86为辅助刺激分子,在免疫细胞活化中不可或缺;MHCⅡ分子检测HLA-DR分子表达,在免疫抗原递呈中起到关键作用。流式细胞仪检测细胞表面成熟标志物,结果见图8。结果表明,与多肽组相比, SARS-CoV2 S蛋白多肽池组的DC细胞CD 1a、CD 11c、CD 80、CD 83和HLA-DR 分别增加了68%、59%、135%、75%和105%。
实施例6DC靶向纳米多肽疫苗刺激MoDC细胞成熟的形态学观察
将从新鲜的抗凝血中分离得到PBMC,经诱导培养6天后获得MoDC细胞。在 MoDC中加入不同的纳米疫苗刺激12h,观察形态变化。如图9所示,MoDC在加入纳米包裹SARS-CoV2多肽疫苗12h后,突触伸长,由刺球状细胞变为多树枝状长突触细胞,有利于对抗原的摄取处理,MoDC由不成熟变为成熟。而未经纳米颗粒包裹的多肽刺激的MoDC细胞形态未发生明显变化,说明细胞还未被刺激成熟。
实施例7利用MTT试验检测纳米靶向多肽疫苗激活DC细胞后对T细胞增殖作用,计算刺激指数SI
结果如图10,刺激指数SI=(实验组OD值-空白组OD值)/(阴性对照OD值- 空白组OD值)计算刺激指数,结果见表1。
表1刺激指数SI
实验发现通过DC靶向纳米多肽疫苗激活后的DC细胞能够刺激T细胞增殖,SI>1,且纳米颗粒包裹S多肽池刺激效果比单肽效果好,使T细胞数显著增加。
实施例8激活后CTL对靶细胞杀伤作用检测——LDH检测法
利用细胞凋亡后LDH释放特性,通过检测靶细胞死亡后产生LDH的量,判定CTL 杀伤能力,结果见图11。结果显示,纳米包裹SARS-CoV2 S蛋白多肽池组对多肽冲击的靶细胞杀伤率最高,达到77%,包裹单肽的纳米疫苗组的杀伤率为20%-25%。实验证明制备的DC靶向纳米多肽疫苗有良好的靶向DC作用,并有效促进CTL杀伤作用,对多肽冲击的T2靶细胞产生强烈的杀伤作用。
以上所述实施方式仅为本发明的优选实施例,而并非本发明可行实施的全部实施例。对于本领域一般技术人员而言,在不背离本发明原理和精神的前提下对其所作出的任何显而易见的改动,都应当被认为包含在本发明的权利要求保护范围之内。
Claims (10)
1.一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,具体包括以下步骤:
步骤S1:称取明胶于烧杯中,加水搅拌溶解,边搅拌边加入丙酮,室温静置,弃去上清液;再加水加温彻底溶解底部沉淀,加入HCl调节pH,再次滴加丙酮,加入用丙酮稀释的戊二醛,室温持续搅拌,挥发丙酮及戊二醛,用双蒸水稀释,使用透析袋,在双蒸水中透析后,在-4℃静置保存;
步骤S2:取步骤S1制备的明胶纳米颗粒于烧杯中,加入甘露糖,NaAc溶液溶解,调节pH,静置一段时间,再持续搅拌2天,透析袋透析24h,4℃保存;
步骤S3:获取SARS-CoV2的S蛋白的氨基酸序列,利用人工神经网络进行S蛋白针对人HLA-A2的MHC-Ⅰ分子结合力预测,筛选亲和力高的表位,并对筛选的表位进行过敏原筛选及理化性质分析,通过对多肽表位进行打分,将筛选出的多肽表位进行过敏原检测,去除可能成为过敏原的多肽表位,确定序列后合成多肽;另外设计一组MHC-Ⅱ分子限制性Th表位的长度为15个氨基酸的多肽表位;
步骤S4:调节明胶纳米颗粒溶液pH,向纳米颗粒中分别加入多肽,4℃震荡4h。
2.如权利要求1所述的一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,所述步骤S1中明胶取0.5g,首次加入10mL双蒸水溶解,再次加入10mL双蒸水50℃彻底溶解。
3.如权利要求1所述的一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,步骤S1中两次丙酮的用量分别为5~30mL,步骤S1中加入1mol/L的HCl调节pH至2.5。
4.如权利要求1所述的一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,步骤S1中加入用2mL丙酮稀释的0.5%的戊二醛。
5.如权利要求1所述的一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,步骤S1与步骤S2中使用40KD的透析袋。
6.如权利要求1所述的一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,步骤S2中取1mg/mL明胶纳米颗粒,0.68g甘露糖使用2mLNaAc溶解。
7.如权利要求1所述的一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,步骤S3中分别利用IEDB网站和NetMHC 4.0Server网站预测I类MHC分子结合位点,和对亲和力进行筛选。
8.如权利要求1所述的一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,步骤S3中采用SYFPEITHI方法进行打分。
9.如权利要求1所述的一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗的制备方法,其特征是,步骤S4中纳米颗粒浓度为1mg/mL,多肽浓度为10g/mL。
10.一种DC细胞靶向的纳米SARS-CoV2 S蛋白多肽池疫苗,其特征是,如所述的步骤S1-S4的制备方法制备。
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