CN114099662A - 一种单核细胞负载e6e7融合蛋白的疫苗组合物及其制备方法与应用 - Google Patents
一种单核细胞负载e6e7融合蛋白的疫苗组合物及其制备方法与应用 Download PDFInfo
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- CN114099662A CN114099662A CN202111254413.9A CN202111254413A CN114099662A CN 114099662 A CN114099662 A CN 114099662A CN 202111254413 A CN202111254413 A CN 202111254413A CN 114099662 A CN114099662 A CN 114099662A
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Abstract
本发明公开了一种单核细胞负载E6E7融合蛋白的疫苗组合物及其制备方法与应用,该疫苗组合物由一定浓度的HPV16 E6E7融合蛋白溶液及单核细胞组成,通过下述方法制备:制备一定浓度的HPV16 E6E7融合蛋白溶液;由外周血或骨髓分离获得单核细胞;将S1获得的一定浓度的HPV16 E6E7融合蛋白溶液与S2获得的一定数量的单核细胞混合,得到单核细胞负载E6E7融合蛋白的疫苗组合物。本发明公开的单核细胞负载E6E7融合蛋白的疫苗组合物及其制备方法与应用能弥补现有技术中的抗原疫苗制备过程繁琐的缺陷。
Description
技术领域
本发明涉及免疫治疗技术领域,具体来说,涉及一种单核细胞负载E6E7融合蛋白的疫苗组合物及其制备方法与应用。
背景技术
全球癌症病例中5%是由人乳头瘤病毒(Human papillomavirus,HPV)引发的,包含宫颈癌、肛门癌、外阴癌、阴道癌、阴茎癌、头颈癌、口咽癌等,其中75.6%的宫颈癌病例是由高危型HPV16/18持续感染引起,肛门癌、外阴癌、阴道癌、阴茎癌、口咽癌患者的HPV感染率分别是88.0%、24.9%、78.0%、50.0%、30.8%。宫颈癌一直高居于18~55岁年龄段女性恶性肿瘤疾病的第二位,在国内每年有约13万宫颈癌病例产生,且发病趋于年轻化。根据WHO/IARC/ICO,HPV Information Centre显示的数据,80-90%以上的女性有一次HPV 感染,80%-90%以上的 HPV 感染可在 2 年内自然清除,4-10%无法自我清除,持续感染发展成为癌前病变(CIN)和癌症。高危型 HPV病毒(HPV 16/18)持续感染,可能造成宫颈癌前病变和宫颈癌、肛门癌、阴茎癌、外阴癌、头颈癌等HPV相关肿瘤。因此,开发一种针对HPV的治疗性疫苗显得尤为必要,但现有技术中的抗原疫苗制备过程繁琐。
发明内容
针对相关技术中的上述技术问题,本发明提出一种单核细胞负载E6E7融合蛋白的疫苗组合物及其制备方法与应用,能够克服现有技术的上述不足。
E6和E7是人体感染HPV病毒之后表达翻译的蛋白,是诱导宫颈癌、肛门癌、阴茎癌、外阴癌、头颈癌等HPV相关肿瘤和癌前病变形成和增殖所必需的蛋白,是HPV相关恶性肿瘤发生和转移的最重要的蛋白分子,且在正常组织不表达,因此E6和E7蛋白成为 HPV疫苗的最理想靶点。同时由于E6和E7这两个蛋白对人体来说属于外源蛋白分子,会被体内免疫系统高效识别和清除。相比以内源性蛋白为靶点的抗肿瘤疫苗产品,E6E7免疫原性更强、所激发的免疫反应更强,有效性更高。近年来研究发现单核细胞可以摄取抗原并直接加工成抗原肽形式在体内激活树突状细胞诱导特异性免疫反应,因而利用单核细胞负载E6E7抗原是值得开发的肿瘤治疗性疫苗形式。
为实现上述技术目的,本发明的技术方案是这样实现的:
一种单核细胞负载E6E7融合蛋白的疫苗组合物,所述疫苗组合物由一定浓度的HPV16 E6E7融合蛋白溶液及单核细胞组成,通过下述方法制备:
S1 制备一定浓度的HPV16 E6E7融合蛋白溶液;
S2 由外周血或骨髓分离获得单核细胞;
S3 将S1获得的一定浓度的HPV16 E6E7融合蛋白溶液与S2获得的一定数量的单核细胞混合,得到单核细胞负载E6E7融合蛋白的疫苗组合物。
优选地,所述HPV16 E6E7融合蛋白溶液的浓度为1mg/mL。
优选地,所述单核细胞采用淋巴分离液Ficoll密度梯度离心法制备。
根据本发明的另一方面,提供了一种单核细胞负载E6E7融合蛋白的疫苗组合物的制备方法,包括以下步骤:
S1 制备一定浓度的HPV16 E6E7融合蛋白溶液;
S2 由外周血或骨髓分离获得单核细胞;
S3 将S1获得的一定浓度的HPV16 E6E7融合蛋白溶液与S2获得的一定数量的单核细胞混合,得到单核细胞负载E6E7融合蛋白的疫苗组合物。
进一步地,S1中制备一定浓度的HPV16 E6E7融合蛋白溶液为采用PBS将HPV16E6E7融合蛋白配制成一定浓度的HPV16 E6E7融合蛋白溶液。
进一步地,S2中由骨髓分离获得单核细胞包括如下步骤:
S21 取小鼠股骨,剪去股骨两头,用PBS反复冲洗股骨骨腔,得到骨髓冲出液,用40μM筛网过滤后,收集至离心管中;
S22 将S21中的离心管离心获得骨髓细胞沉淀;加入PBS重悬骨髓细胞;将骨髓细胞悬液加入含Ficoll淋巴分离液的离心管中;
S23 将S2中的离心管离心,用吸管吸取第二层环状乳白色单核细胞层到另一离心管中,补加PBS,离心;
S24 弃上清,加PBS重悬,再离心,弃上清,得到单核细胞团块,用适量含10%灭活FBS的RPMI 1640培养基重悬,并计数。
进一步地,S3中将HPV16 E6E7融合蛋白溶液与单核细胞混合包括以下步骤:
S31 将HPV16 E6E7融合蛋白溶液溶解于含10%灭活FBS的RPMI 1640培养基中,使其浓度为1mg/mL;
S32 将单核细胞加入到所述HPV16 E6E7融合蛋白溶液中,保证单核细胞密度一致,均为2×107 个细胞/mL;
S33 将步骤S32的细胞悬液放入恒温培养箱中,37℃,5% CO2孵育2h,每半小时振荡1次;
S34 孵育结束后,将细胞悬液离心,弃上清,再加入适量PBS重悬,再离心,弃上清,得到负载HPV16 E6E7融合蛋白溶液抗原的单核细胞团块;
S35 负载HPV16 E6E7融合蛋白溶液抗原的单核细胞用RPMI 1640培养基重悬,使细胞终浓度为5×107 个细胞/mL,得到疫苗组合物。
本发明还提供了一种上述疫苗组合物在制备用于治疗HPV16 阳性肿瘤制品中的应用。
进一步地,所述疫苗组合物采用静脉注射的方式。
本发明的有益效果:本发明提供了一种新的HPV16 E6E7融合蛋白溶液抗原疫苗组合物及其制备方法,通过简化的单核细胞分离操作减少了常规单核细胞分离及分化产生DC的操作步骤,极大程度地降低了疫苗制备成本;直接利用分离的单核细胞负载HPV16 E6E7融合蛋白溶液抗原,能达到显著的抗击肿瘤效果,为更广泛的临床试验提供了前期技术基础。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是根据本发明实施例所述的小鼠骨髓来源的单核细胞吞噬OVA-FITC的效率检测结果,包括流式代表性图和统计数据条形图;
图2是根据本发明实施例所述的单核细胞HPV16 E6E7蛋白溶液肿瘤疫苗免疫C57BL/6受体鼠后第14天外周血抗体的检测结果;
图3是根据本发明实施例所述的单核细胞HPV16 E6E7融合蛋白溶液肿瘤疫苗免疫C57BL/6受体鼠后第14天外周血特异性T细胞的检测结果,包括流式代表性图和统计数据图;
图4是根据本发明实施例所述的HPV16 E6E7融合蛋白溶液单核细胞疫苗对TC-1细胞系荷瘤小鼠肿瘤生长曲线(预防性模型),在第-21、-14、-7天注射疫苗,第0天接种肿瘤细胞;
图5是根据本发明实施例所述的HPV16 E6E7融合蛋白溶液单核细胞疫苗对TC-1细胞系荷瘤小鼠肿瘤生长曲线(治疗性模型),在第0天接种肿瘤细胞,第7、14天注射疫苗。
具体实施方式
以下的实施例用于为本领域中的普通技术人员提供有关如何实施和使用本发明的完整披露和描述,并且这些例子并非意在对发明人所认为的发明范围进行限制,亦非意指下文的实验是被实施的全部实验而且是仅可实施的实验。实施例中未注明具体条件的试剂和设备,通常为本技术领域常规市购的试剂、设备;实施例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件。
具体地,关于下面实施例中涉及的原料生产厂家如表1所示:
表1. 原料表
下面结合附图和具体实施方式进一步详细说明本发明。
实施例1
单核细胞的分离,包括如下步骤:
(1)颈脱臼法处死小鼠,取其2只股骨,剪去股骨两头,用注射器吸入适量PBS反复冲洗股骨骨腔,得到骨髓冲出液,用40μM筛网过滤后,收集至PE离心管中。
(2)300-500g离心5min,获得骨髓细胞沉淀;加入4 mL PBS重悬骨髓细胞;同时在另一PE离心管中加入3mL Ficoll淋巴分离液。将骨髓细胞悬液轻轻加入含Ficoll淋巴分离液的PE离心管中,注意不要破坏细胞悬液与淋巴分离液的接触界面。
(3)将PE离心管400g离心30min,因密度不同离心管中由上到下分为四层:第一层为血浆层,第二层为环状乳白色单核细胞层,第三层为透明分离液层,第四层为红细胞层,用吸管小心吸取第二层环状乳白色单核细胞层到另一50ml离心管中,补加PBS,400g离心5~10min。
(4)弃上清,加PBS重悬,400g离心5~10min,弃上清,得到单核细胞团块,适量含10%灭活FBS的RPMI 1640培养基重悬,并计数。
实施例2
将实施例1获得的单核细胞负载HPV16 E6E7融合蛋白溶液或OVA-FITC抗原,包括如下步骤:
(1)将HPV16 E6E7融合蛋白或OVA-FITC溶解于含10%灭活FBS的RPMI 1640培养基中,使其浓度为1mg/mL。HPV16 E6蛋白的氨基酸序列如下:MHQKRTAMFQ DPQERPRKLPQLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIV YRDGNPYAVC DKCLKFYSKI SEYRHYCYSLYGTTLEQQYN KPLCDLLIRC INCQKPLCPE EKQRHLDKKQ RFHNIRGRWT GRCMSCCRSS RTRRETQL(158aa)(SEQ ID NO:1);HPV16 E7蛋白的氨基酸序列如下:MHGDTPTLHE YMLDLQPETTDLYCYEQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIVCPICSQKP(98aa)(SEQ ID NO:2)。
(2)将单核细胞加入到上述HPV16 E6E7融合蛋白溶液中,保证单核细胞密度一致,均为2×107 个细胞/mL。
(3)将步骤(2)的细胞悬液放入恒温培养箱中,37℃,5% CO2孵育2h,每半小时振荡1次。
(4)孵育结束后,将细胞悬液以400g离心5~10min,弃上清,再加入适量PBS重悬,再按400g离心5~10min,弃上清,得到负载HPV16 E6E7融合蛋白溶液抗原的单核细胞团块。
(5)上述负载HPV16 E6E7融合蛋白溶液抗原的单核细胞用RPMI 1640培养基重悬,使细胞终浓度为5×107 个细胞/mL,该混合液即疫苗组合物。用流式细胞仪检测单核细胞负载HPV16 E6E7融合蛋白溶液或OVA-FITC情况,结果如图1所示。
实施例3
将实施例2获得的疫苗组合物免疫小鼠模型,包括如下步骤:
(1)将负载HPV16 E6E7融合蛋白溶液的单核细胞悬液按200 μL的体积由尾静脉注射入为C57BL/6小鼠体内,每只小鼠接种细胞量为1×107,对照组小鼠注射等体积PBS。连续接种3次,每隔7天接种1次。
(2)肿瘤接种后第7天开始,连续追踪测量小鼠肿瘤生长状况,并于第一次接种疫苗第14天分析血清中抗体产生情况,结果如图2-3所示。
(3)预防性模型如图4所示:距第一次接种疫苗第14天,对每只小鼠接种肿瘤细胞。每只小鼠肿瘤细胞接种量为1×106,接种方式为皮下注射。
(4)治疗性模型如图5所示:每只小鼠肿瘤细胞接种量为1×106,接种方式为皮下注射。分别在肿瘤接种后第7、14天接种HPV16 E6E7融合蛋白溶液单核细胞疫苗。
该疫苗组合物具有特异性强的高效的抵抗作用,能应用在制备用于治疗实体肿瘤制品中。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 山西协策企业管理咨询有限公司
<120> 一种单核细胞负载E6E7融合蛋白的疫苗组合物及其制备方法与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 158
<212> PRT
<213> Human papillomavirus type 16
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<212> PRT
<213> Human papillomavirus type 16
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Claims (9)
1. 一种单核细胞负载E6E7融合蛋白的疫苗组合物,其特征在于,所述疫苗组合物由一定浓度的HPV16 E6E7融合蛋白溶液及单核细胞组成,通过下述方法制备:
S1 制备一定浓度的HPV16 E6E7融合蛋白溶液;
S2 由外周血或骨髓分离获得单核细胞;
S3 将S1获得的一定浓度的HPV16 E6E7融合蛋白溶液与S2获得的一定数量的单核细胞混合,得到单核细胞负载E6E7融合蛋白的疫苗组合物。
2. 根据权利要求1所述的疫苗组合物,其特征在于,所述HPV16 E6E7融合蛋白溶液的浓度为1mg/mL。
3.根据权利要求1所述的疫苗组合物,其特征在于,所述单核细胞采用淋巴分离液Ficoll密度梯度离心法制备。
4.一种单核细胞负载E6E7融合蛋白的疫苗组合物的制备方法,其特征在于,包括以下步骤:
S1 制备一定浓度的HPV16 E6E7融合蛋白溶液;
S2 由外周血或骨髓分离获得单核细胞;
S3 将S1获得的一定浓度的HPV16 E6E7融合蛋白溶液与S2获得的一定数量的单核细胞混合,得到单核细胞负载E6E7融合蛋白的疫苗组合物。
5. 根据权利要求4所述的疫苗组合物的制备方法,其特征在于,S1中制备一定浓度的HPV16 E6E7融合蛋白溶液为采用PBS将HPV16 E6E7融合蛋白配制成一定浓度的HPV16 E6E7融合蛋白溶液。
6.根据权利要求4所述的疫苗组合物的制备方法,其特征在于,S2中由骨髓分离获得单核细胞包括如下步骤:
S21 取小鼠股骨,剪去股骨两头,用PBS反复冲洗股骨骨腔,得到骨髓冲出液,用40μM筛网过滤后,收集至离心管中;
S22 将S21中的离心管离心获得骨髓细胞沉淀;加入PBS重悬骨髓细胞;将骨髓细胞悬液加入含Ficoll淋巴分离液的离心管中;
S23 将S2中的离心管离心,用吸管吸取第二层环状乳白色单核细胞层到另一离心管中,补加PBS,离心;
S24 弃上清,加PBS重悬,再离心,弃上清,得到单核细胞团块,用适量含10%灭活FBS的RPMI 1640培养基重悬,并计数。
7. 根据权利要求4所述的疫苗组合物的制备方法,其特征在于,S3中将HPV16 E6E7融合蛋白溶液与单核细胞混合包括以下步骤:
S31 将HPV16 E6E7融合蛋白溶液溶解于含10%灭活FBS的RPMI 1640培养基中,使其浓度为1mg/mL;
S32 将单核细胞加入到所述HPV16 E6E7融合蛋白溶液中,保证单核细胞密度一致,均为2×107 个细胞/mL;
S33 将步骤S32的细胞悬液放入恒温培养箱中,37℃,5% CO2孵育2h,每半小时振荡1次;
S34 孵育结束后,将细胞悬液离心,弃上清,再加入适量PBS重悬,再离心,弃上清,得到负载HPV16 E6E7融合蛋白溶液抗原的单核细胞团块;
S35 负载HPV16 E6E7融合蛋白溶液抗原的单核细胞用RPMI 1640培养基重悬,使细胞终浓度为5×107 个细胞/mL,得到疫苗组合物。
8. 一种如权利要求1所述的疫苗组合物在制备用于治疗HPV16 阳性肿瘤制品中的应用。
9.根据权利要求8所述的应用,其特征在于,所述疫苗组合物采用静脉注射的方式。
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