CN112957461B - 一种形貌可控自佐剂宫颈癌多肽疫苗的制备及性能表征方法 - Google Patents
一种形貌可控自佐剂宫颈癌多肽疫苗的制备及性能表征方法 Download PDFInfo
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Abstract
一种形貌可控自佐剂宫颈癌多肽疫苗的制备及性能表征方法。基于4‑氨基脯氨酸五肽的结构特征,合成了HPV E7抗原肽功能化的五肽衍生物,通过共组装制备得到。同时,将4‑氨基脯氨酸替换成天然脯氨酸,通过共组装制备纳米颗粒多肽疫苗,考察疫苗形貌对疫苗免疫应答的影响。生物实验表明多肽疫苗能够成功诱导树突成熟和抗原呈递并促进T细胞增殖,具有自佐剂功能。细胞摄取实验结果表明纳米纤维多肽疫苗比纳米颗粒多肽疫苗在细胞内具有更长的保留时间,而且其在促进DC成熟,引流淋巴结,T淋巴细胞浸润肿瘤组织以及最终杀伤肿瘤细胞方面具有更优异的性能。与抗PD‑1抗体组合使用,证明了组合治疗策略可以提高纳米纤维肽疫苗治疗效果。
Description
技术领域
本发明涉及多肽纳米疫苗技术领域,具体涉及一种形貌可控的免疫增强型治疗HPV相关癌症的多肽疫苗的制备方法。
技术背景
免疫疗法已被证明是一种有前途的癌症治疗策略,因为它能够引起宿主被抑制的天然免疫反应,以防御和根除癌细胞。在过去的几十年中,通过利用诸如癌症疫苗,免疫佐剂,细胞因子或免疫检查点阻断剂之类的疗法来激发内部免疫细胞或直接利用工程化的嵌合抗原受体T细胞疗法,证实了基于该策略的癌症免疫疗法的治疗效果。在这些免疫疗法中,由源自病原体的抗原组成的癌症疫苗不仅能够引发免疫反应以清除已存在的癌细胞,还可以建立免疫记忆来防御进一步的感染。迄今为止,基于纳米药物递送平台的优势,已经开发了无机纳米颗粒,聚合物平台,脂质体和树状聚合物等多种材料用于制备癌症疫苗,以提高其激发免疫应答的能力。尽管癌症疫苗具有巨大的治疗潜力,但由于低免疫应答以及潜在的不良副作用(例如细胞因子释放综合征)一些癌症疫苗的临床应用受到严重阻碍。
短肽由有限数量的氨基酸残基组成,由于其出色的生物相容性和组装能力,已被广泛用于组织再生,药物递送和癌症疫苗等多个领域。在开发癌症疫苗方面,多肽可以直接与免疫疗法一起使用,也可以自组装成形态明确的纳米结构作为抗原的递送平台或免疫佐剂。与抗体相比,肽抗原易于合成,并且在稳定性和防止由病原体污染物引起的过敏或自身免疫反应方面显示出显着优势。此外,由于多肽之间可靠的非共价相互作用,短肽自组装成纳米结构或水凝胶可负载和递送其他免疫治疗剂。将具有组装倾向的肽序列与肽抗原结合,可以产生无需常规佐剂(如铝佐剂)的自佐剂疫苗。在这些多肽疫苗中,已证明疫苗的适应性免疫反应与多肽组装体密切相关。但是,多肽纳米结构的形貌对抗原疫苗的影响研究较少,因此限制了有效多肽疫苗的开发。为了解决这个问题,在此报道了形貌可控的自佐剂多肽疫苗,并阐明了纳米纤维多肽疫苗的更优异的免疫应答能力,可有效抑制与HPV相关的肿瘤。
发明内容
本发明目的是研究多肽纳米结构的形貌对抗原疫苗的影响,提供形貌可控的免疫增强型自佐剂多肽疫苗制备方法,用于HPV相关癌症的有效治疗。该多肽疫苗制备方法简单,反应条件温和,操作简便。
为实现上述目的,本发明提供的技术方案为:
一种形貌可控自佐剂宫颈癌多肽疫苗的制备方法,
多肽AmpF,PF,E749-57,E744-57,AmpFE749-57,AmpFE744-57,PFE749-57和PFE744-57是通过标准Fmoc固相肽合成(SPPS)方法合成的。在制备多肽疫苗之前,首先将多肽冻干粉(AmpF,PF,AmpFE749-57,AmpFE744-57,PFE749-57和PFE744-57)溶解在水中来制备浓度为5mM的不同多肽的储备溶液。多肽疫苗AmpF-E749-57和AmpF-E744-57是通过将多肽AmpF和AmpFE749-57或多肽AmpF和AmpFE744-57的溶液以90:10的摩尔比混合制备的,最终的总浓度为2mM。同时,按照相同的方案,以90:10的摩尔比混合多肽PF和PFE749-57或PF和PFE744-57的溶液制备多肽疫苗PF-E749-57和PF-E744-57。同时,还制备了浓度为2mM的AmpF,PF,E749-57和E744-57的多肽溶液。在进行所有表征和生物学实验之前,将多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57的溶液均放置2天。在研究多肽疫苗自组装之前,通过记录尼罗红的最大发射波长来估算多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57,PF-E744-57的临界聚集浓度(CAC)。研究表明,多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57聚集浓度均低于10μM。
本发明制备得到的多肽疫苗的二级结构通过圆二色(CD)光谱进行了表征。多肽疫苗AmpF-E749-57和AmpF-E744-57的CD光谱分别在193和215nm或195和213nm处显示最小或最大强度。这些信号表明多肽疫苗AmpF-E7初步形成了β-折叠构象。此外,多肽疫苗PF-E749-57和PF-E744-57的CD光谱在200和220nm处显示两个正峰,表明多肽疫苗PF-E7采用无规卷曲构象。硫黄素T(ThT)分析证实了由疫苗AmpF-E7形成的β-折叠。添加疫苗AmpF-E749-57和AmpF-E744-57后,ThT荧光发射强度分别增加了230倍和240倍。值得注意的是,多肽疫苗诱导的ThT荧光强度的增加与仅由多肽AmpF诱导的ThT荧光强度的增加相当,这意味着长抗原表位不参与AmpF结构域的自组装。相反,在ThT溶液中添加PF-E7不会引起ThT荧光的明显改善,这证实了多肽疫苗PF-E7形成了无规卷曲。
通过原子力显微镜(AFM)和透射电子显微镜(TEM)研究了多肽疫苗的形貌。AFM图像显示多肽疫苗AmpF-E749-57和AmpF-E744-57为纤维状,单根纤维的高度约为3nm。TEM图像证实了由多肽疫苗AmpF-E7形成的纳米纤维,单根纳米纤维的宽度约为3nm,这与AFM图像中显示的纳米纤维的高度一致。相反,AFM和TEM研究均显示,多肽疫苗PF-E749-57和PF-E744-57分别形成平均直径为100±20nm和100±10nm的纳米颗粒。
本发明的优点和有益效果:
(1)本发明所形成的多肽组装体具有生物兼容性、低免疫原性和生物降解性等优点。(2)本发明所有的反应条件非常温和,制备方法简单,操作简便。(3)本发明得到的多肽疫苗具有自佐剂功能,尤其是纤维状多肽疫苗具有更强的免疫增强功能,能够有效抑制HPV相关肿瘤生长。(4)本发明证实了纤维状多肽疫苗由于细胞内更长时间的滞留时间比纳米颗粒状多肽疫苗具有更好的免疫增强功能,对高效多肽疫苗的设计具有指导意义。(5)本发明制备的多肽疫苗与免疫检查点抑制剂抗PD-1抗体联合使用可以进一步增强免疫治疗效果,更有效的抑制肿瘤生长。
附图说明
图1.五肽AmpF和PF,抗原肽E749-57和E744-57,含抗原表位的肽AmpFE749-57,AmpFE744-57,PFE749-57和PFE744-57,FAM标记的肽AmpF-FAM,PF-FAM,E749-57-FAM和E744-57-FAM的化学结构式。
图2.在多肽疫苗AmpF-E749-57(a),AmpF-E744-57(b),PF-E749-57(c)和PF-E744-57(d)中的尼罗红的最大荧光发射波长(λmax)与多肽浓度之间的函数曲线图(0.01至50μM)。
图3.(a和b)在多肽AmpF和PF(a)或肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57(b)中的ThT溶液的荧光光谱图。(c和d)在多肽AmpF和PF(c)或肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57(d)中的ThT分子在最大发射(@483nm)处的荧光强度。
图4.(a)多肽疫苗AmpF-E7(AmpF-E749-57和AmpF-E744-57)和PF-E7(PF-E749-57和AmpF-E744-57)的制备示意图。(b)多肽疫苗AmpF-E7和PF-E7在pH 7.4下的CD光谱。(c-j)多肽疫苗在pH 7.4下的AFM和TEM图像,AmpF-E749-57(c,g),AmpF-E744-57(d,h),PF-E749-57(e,i)和PF-E744-57(f,j)。插图显示所选纳米纤维的高度或相应纳米粒子的直径分布。
图5.在五肽AmpF和PF,多肽疫苗AmpF-E749-57,PF-E749-57,AmpF-E744-57和PF-E744-57,肽抗原E749-57和E744-57的存在下培养的3T3,TC-1和DC2.4细胞的细胞活力(1:AmpF;2:PF;3:AmpF-E749-57;4:PF-E749-57;5:E749-57;6:AmpF-E744-57;7:PF-E744-57;8:E744-57)。
图6.(a,b)与FAM标记的五肽,游离抗原肽或多肽疫苗共孵育2、4、8、12、16、24小时后BMDC的流式细胞仪分析结果(a)和DC2.4细胞的CLSM图像(b)。(c)对BMDC流式细胞仪分析结果的量化。(d,e)与疫苗AmpF-E7和PF-E7共孵育2、4和8h的DC2.4细胞的CLSM图像(d)和LysoTracker与FAM信号之间的皮尔逊相关系数(e)。蓝色:被DAPI染色的细胞核;绿色,FAM标记的多肽;红色,溶酶体荧光探针。比例尺:40μm。0:PBS,1:AmpF,2:PF,3:AmpF-E749-57,4:PF-E749-57,5:E749-57,6:AmpF-E744-57,7:PF-E744-57,8:E744-57。
图7.(a)多肽疫苗刺激骨间充质干细胞(MSC)衍生的BMDC成熟以及体外CTL诱导TC-1细胞裂解的示意图。(b-d)CD86+和CD80+(b),MHC-I(c)和MHC-II(d)在五肽AmpF和PF,游离抗原E749-57和E749-57或多肽疫苗AmpF-E7和PF-E7刺激的BMDC上的表达。(e-h)用CFSE标记的T淋巴细胞的流式细胞分析结果(e)和定量统计图(f),或将经不同多肽或疫苗预处理的BMDC与脾中T淋巴细胞共孵育后,T淋巴细胞中CD8+T细胞(g)和CD4+T细胞(h)定量统计图。(i)经预处理后的BMDC激活的T淋巴细胞与TC-1细胞共孵育后,TC-1的细胞活性定量统计。
图8.(a,c)皮下注射FAM标记的多肽,多肽疫苗和游离多肽抗原的小鼠在不同时间时体内荧光图像及荧光强度定量统计。(b,d)从注射了FAM标记的多肽,多肽疫苗和游离多肽抗原的小鼠中解剖出的脾脏(顶部)和LNs(底部)的离体荧光图像及荧光强度定量统计。(e-f)皮下注射FAM标记的多肽,多肽疫苗和游离多肽抗原48小时后的小鼠解剖出的LNs中CD4+(e)和CD8+(f)T细胞的百分比。
图9.(a)对HPV相关的TC-1肿瘤模型小鼠多肽疫苗免疫治疗的示意图。(b)用五肽,抗原肽E7,多肽疫苗AmpF-E7和PF-E7治疗后,小鼠的相对肿瘤大小。(c)在使用不同治疗21天后TC-1肿瘤的代表性图像。(d)采用不同治疗方法后的小鼠的体重。(e)对浸润到肿瘤组织中的CD4+和CD8+T细胞的流式细胞分析结果图。(f,g)对浸润到肿瘤组织中的CD4+和CD8+T细胞的定量统计图。
图10.(a,b)经五肽,游离抗原肽E7,多肽疫苗AmpF-E7和PF-E7处理的小鼠的脾脏中的CD4+和CD8+T细胞的定量统计图。(c)不同处理的小鼠的LNs中的TCM(CD44HiCD62L+)细胞的定量统计图。(d-e)不同处理的小鼠脾脏中的CD4+(d)或CD8+(d)T细胞相关的细胞因子IFN-γ+的定量统计图。(f-g)不同处理的小鼠眼眶血液中细胞因子IL-2(f)和TNF-α(g)的ELISA试剂盒分析结果图。(h-j)对不同处理的小鼠的肿瘤组织的H&E(h),抗CD4+(i)或抗CD8+(j)免疫组织化学(IHC)染色的代表性图像。比例尺:50μm。
图11.(a)对HPV相关的TC-1肿瘤模型小鼠多肽疫苗和抗PD-1抗体联合免疫治疗的示意图。(b)用PBS,抗PD-1,抗原E744-57+抗PD-1,多肽疫苗PF-E744-57+抗PD-1或多肽疫苗AmpF-E744-57+抗PD-1治疗后,小鼠的相对肿瘤大小。(c)在使用不同治疗21天后TC-1肿瘤的代表性图像。(d)采用不同治疗方法后的小鼠的体重。(e)对浸润到肿瘤组织中的CD4+和CD8+T细胞的流式细胞分析结果图。(f,g)对浸润到肿瘤组织中的CD4+和CD8+T细胞的定量统计图。
(h-j)不同处理的小鼠眼眶血液中细胞因子TNF-α(h),IL-2(i)和TNF-α(j)的ELISA试剂盒分析结果图。0:PBS;9:抗PD-1;10:E744-57+抗PD-1;11:PF-E744-57+抗PD-1;12:AmpF-E744-57+抗PD-1。(k-m)对不同处理的小鼠的肿瘤组织的H&E(k),抗CD4+(l)或抗CD8+(m)免疫组织化学(IHC)染色的代表性图像。比例尺:50μm。
具体实施方式
下面将通过实例描述,阐述本发明的优点和效果。
多肽疫苗AmpF-E7和PF-E7的合成与表征
图1为本发明实施例中所有多肽的结构式。如图2所示,在研究多肽疫苗自组装之前,通过记录尼罗红的最大发射波长来估算多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57,PF-E744-57的临界聚集浓度(CAC)。本发明基于尼罗红最大发射波长随着所处微环境疏水性变化而变化的原理,尼罗红被用作荧光探针,将尼罗红乙醇溶液(2μL 100μM)加入到不同浓度的每种多肽(2mL)中,并孵育过夜以进行测量。设置激发波长为550nm,激发和发射光源的狭缝宽度为10nm,用荧光分光光度计(Agilent Cary Eclipse)记录600至700nm波长范围内尼罗红的荧光光谱。将尼罗红的最大荧光发射波长绘制为多肽浓度的函数,以估算CAC值。研究表明,多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57聚集浓度均低于10μM。
本发明多肽AmpF,PF,E749-57,E744-57,AmpFE749-57,AmpFE744-57,PFE749-57和PFE744-57是通过标准Fmoc固相肽合成(SPPS)方法合成的。在制备多肽疫苗之前,首先将多肽冻干粉(AmpF,PF,AmpFE749-57,AmpFE744-57,PFE749-57和PFE744-57)溶解在水中来制备浓度为5mM的不同多肽的储备溶液。多肽疫苗AmpF-E749-57和AmpF-E744-57是通过将多肽AmpF和AmpFE749-57或多肽AmpF和AmpFE744-57的溶液以90:10的摩尔比混合制备的,最终的总浓度为2mM。同时,按照相同的方案,以90:10的摩尔比混合多肽PF和PFE749-57或PF和PFE744-57的溶液制备多肽疫苗PF-E749-57和PF-E744-57。同时,还制备了浓度为2mM的AmpF,PF,E749-57和E744-57的多肽溶液。在进行所有表征和生物学实验之前,将多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57的溶液均放置2天。
多肽疫苗的二级结构通过圆二色(CD)光谱进行了表征(图4b)。多肽疫苗AmpF-E749-57和AmpF-E744-57的CD光谱分别在193和215nm或195和213nm处显示最小或最大强度。这些信号表明多肽疫苗AmpF-E7初步形成了β-折叠构象。此外,多肽疫苗PF-E749-57和PF-E744-57的CD光谱在200和220nm处显示两个正峰,表明多肽疫苗PF-E7采用无规卷曲构象。硫黄素T(ThT)分析证实了由疫苗AmpF-E7形成的β-折叠(图2)。添加疫苗AmpF-E749-57和AmpF-E744-57后,ThT荧光发射强度分别增加了230倍和240倍。值得注意的是,多肽疫苗诱导的ThT荧光强度的增加与仅由多肽AmpF诱导的ThT荧光强度的增加相当,这意味着长抗原表位不参与AmpF结构域的自组装。相反,在ThT溶液中添加PF-E7不会引起ThT荧光的明显改善,这证实了多肽疫苗PF-E7形成了无规卷曲。
圆二色性(CD)光谱表征中,五肽AmpF,PF,多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57,肽抗原E749-57和E744-57的CD光谱通过分光光度计记录(Biologic MOS-500)。将浓度为2mM的多肽肽溶液转移到两片0.1mm的石英载玻夹片中间进行扫描。波长扫描范围为190nm至250nm,间隔为1.0nm。
硫黄素T(ThT)结合测定中,使用荧光分光光度计(Agilent Cary Eclipse)记录在多肽存在或不存在条件下ThT的荧光光谱。将ThT(20μM)加入多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57(2mM)中放置过夜,然后用于测量。在测量期间,使用光程为1cm的石英池,设置激发波长为421nm,激发和发射光源的狭缝宽度为20nm,记录以450至600nm范围内的荧光光谱。
通过原子力显微镜(AFM)和透射电子显微镜(TEM)研究了多肽疫苗的形貌(图4,c-j)。AFM图像显示多肽疫苗AmpF-E749-57和AmpF-E744-57为纤维状,单根纤维的高度约为3nm(图4,c和d)。TEM图像证实了由多肽疫苗AmpF-E7形成的纳米纤维,单根纳米纤维的宽度约为3nm(图4,g和h),这与AFM图像中显示的纳米纤维的高度一致。相反,AFM和TEM研究均显示,多肽疫苗PF-E749-57和PF-E744-57分别形成平均直径为100±20nm和100±10nm的纳米颗粒(图4,e,f,i和j)。
原子力显微镜(AFM)研究中,在敲击模式下,使用Bruker ICON仪器记录多肽疫苗的AFM图像。首先,将10μL浓度为2mM的多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57滴加在云母片表面,静置5分钟。残留的液体用滤纸吸干,在空气中晾干后用于测试。
透射电子显微镜(TEM)研究中,多肽疫苗的TEM图像是在Philips Tecnai G220 S-TWIN显微镜上获得的。将多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57(2mM,10μL)滴加到碳涂层铜网格的表面并静置5分钟。用滤纸将溶液吸干,随后将10μL的2wt%乙酸双氧铀滴加在铜网表面,静置3分钟后用滤纸将其除去。在测试之前,将样品存储在干燥器中。
BMDCs和DC2.4细胞对多肽疫苗的细胞摄取
进一步通过甲基噻唑基四唑鎓(MTT)分析评估了多肽疫苗AmpF-E7和PF-E7对APC,健康细胞和肿瘤细胞(包括DC2.4,3T3细胞和TC-1细胞)的细胞毒性(图5)。用浓度范围从0到200μM的多肽疫苗处理所有这些细胞,48小时所有细胞的细胞活力的降低可忽略不计,这表明多肽疫苗AmpF-E7和PF-E7具有出色的生物相容性。随后,利用流式细胞仪和激光共聚焦扫描显微镜(CLSM)对骨髓树突状细胞(BMDC)和DC2.4细胞进行了多肽疫苗的细胞摄取实验(图6)。流式细胞仪分析显示所有多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57逐渐被BMDC摄取(图6a)。荧光强度定量分析显示,所有处理组荧光强度约在8小时后达到最大。与游离肽抗原E744-57和E749-57相比,BMDC对多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57的摄取明显增强,表明该多肽疫苗组装体可以促进细胞摄取。值得注意的是,共孵育12小时后,纳米纤维肽疫苗AmpF-E749-57和AmpF-E744-57在BMDC中的残留量显著高于纳米颗粒疫苗PF-E749-57和PF-E744-57。直到共孵育24小时,BMDC中多肽疫苗AmpF-E749-57和AmpF-E744-57的荧光强度的下降量可忽略不计,而多肽疫苗PF-E749-57和PF-E744-57的荧光强度几乎下降到游离抗原肽的水平(图6a,c)。通过DC2.4细胞的CLSM实验,证实了多肽疫苗的细胞摄取(图6b),与流式细胞仪检测结果一致。将流式细胞仪与CLSM实验结果均表明,与纳米颗粒疫苗和游离抗原肽相比,纳米原纤维肽疫苗在细胞中的保留时间更长,这证明了形态学调控在优化疫苗输送效率中的关键作用,并可能有助于增强疫苗的免疫反应。
利用CLSM监测多肽疫苗在细胞中的定位验证多肽疫苗的细胞摄取途径(图6,d和e)。用红色的Lyso-Tracker标记细胞溶酶体,通过绿色的FAM标记的多肽疫苗与红色溶酶体的荧光信号的Pearson相关系数,可以确定多肽疫苗的摄取途径。在整个摄取过程中游离肽抗原E744-57和E749-57的Pearson相关系数较小,但纳米原纤维和纳米颗粒多肽疫苗在与DC2.4细胞共孵育2小时后均显示出最大的相关系数。这些结果表明多肽疫苗通过溶酶体介导的内吞作用进入细胞。延长DC2.4细胞的孵育时间会导致共定位相关系数降低,这表明多肽疫苗可以成功实现溶酶体逃逸。
多肽疫苗诱导的抗原呈递和T细胞增殖
为了验证所制备的自佐剂多肽疫苗的免疫治疗潜力,对用多肽疫苗AmpF-E7和PF-E7处理后的BMDC进行了流式细胞术分析,研究了BMDC的抗原呈递和其对脾细胞中T细胞的增殖的影响(图7)。流式细胞分析结果显示,多肽疫苗AmpF-E749-57,AmpF-E744-57,PF-E749-57和PF-E744-57处理的BMDC表面CD86+,CD80+,MHC-I和MHC-II的表达明显比五肽AmpF和PF以及游离抗原肽E749-57和E744-57处理组BMDC表面的表达高(图7,b,c,d)。这些结果表明BMDC能够被纳米纤维和纳米颗粒多肽疫苗诱导成熟并实现抗原呈递,从而证明了多肽疫苗具有自佐剂功能。由纳米纤维多肽疫苗AmpF-E7诱导的BMDC表面促成熟及抗原呈递特征分子的百分比明显高于纳米颗粒多肽疫苗。这一发现表明,与纳米颗粒多肽疫苗相比,纳米纤维多肽疫苗更有利于BMDC的成熟和抗原呈递。
通过流式细胞术进一步研究了与被不同多肽疫苗处理后的BMDC共孵育后的T淋巴细胞的增殖情况(图7,e-h)。与五肽AmpF和PF以及游离抗原肽E749-57和E744-57处理组相比,与多肽疫苗AmpF-E7和PF-E7预处理的BMDC共孵育后,原始CFSE标记的T淋巴细胞显著分裂增殖。特别是,用纳米纤维疫苗AmpF-E7预处理后的BMDC诱导T细胞增殖的能力更出色。此外,与多肽疫苗预处理的BMDC共孵育的T淋巴细胞中的CD8+和CD4+T细胞的百分比比其他预处理组更高。同时,纳米纤维疫苗诱导增殖的T淋巴细胞中CD8+和CD4+T细胞的百分比约为纳米颗粒多肽疫苗的1.5倍和1.4倍。该结果证实了与纳米颗粒多肽疫苗相比,纳米纤维多肽疫苗更促进了T细胞的增殖。
进一步研究了多肽疫苗在体外诱导CTL的产生并进一步杀伤TC-1肿瘤细胞的能力(图7i)。经五肽或游离肽抗原预处理的BMDC孵育的T淋巴细胞存在下,TC-1细胞的死亡率几乎可以忽略不计。同样,用纳米纤维疫苗AmpF-E7激活的T淋巴细胞处理的TC-1细胞的死亡率远高于与纳米颗粒多肽疫苗PF-E7处理组。这些结果表明,通过多肽疫苗预处理的BMDCs促进了T淋巴细胞增殖并促进了肿瘤细胞死亡,肿瘤细胞死亡率与多肽疫苗的形貌密切相关。
多肽疫苗的淋巴结富集和T细胞增殖
由于引流淋巴结在引发免疫反应中的关键作用,在小鼠尾根部皮下注射FAM标记的多肽疫苗后,评估了不同时间后小鼠的LNs上多肽疫苗的积累(图8)。注射多肽疫苗AmpF-E7和PF-E7的小鼠的荧光图像在LNs处出现了明显的荧光信号,而用游离肽抗原处理的小鼠在LNs处却没有看到明显的荧光信号(图8a)。从注射了多肽疫苗AmpF-E7和PF-E7的小鼠身上解剖出的LNs和脾脏的离体图像也显示出较强的荧光信号(图8b)。这些结果清楚地表明,多肽疫苗在LN和脾脏中的有效积累,可能归因于成熟BMDC或单独疫苗的迁移。不同处理组小鼠体内或离体荧光强度的定量分析结果显示,用纳米纤维肽疫苗AmpF-E749-57和AmpF-E744-57处理12小时的小鼠的LNs上荧光信号强度是PF-E749-57和PF-E744-57疫苗处理组的3-4倍。同时,在注射多肽疫苗48小时后,纳米纤维疫苗处理组的LNs和脾仍有可见的荧光信号,这表明纳米纤维疫苗在LN和脾中具有更长的保留时间。
还通过流式细胞术分析了在48小时后不同处理组的小鼠解剖的LNs中CD4+和CD8+T细胞的增殖情况(图8e和f)。发现,只有用纳米纤维疫苗AmpF-E744-57和AmpF-E749-57处理组的LNs中表现出CD4+和CD8+T细胞的明显增殖,表明与纳米颗粒多肽疫苗相比,纳米纤维疫苗更有利于诱导T细胞增殖。
多肽疫苗的HPV相关肿瘤免疫治疗
基于多肽疫苗在抗原呈递,LNs富集和滞留以及引发T细胞相关免疫应答方面的发现,通过对小鼠皮下注射HPV相关TC-1肿瘤细胞研究了多肽疫苗在肿瘤免疫治疗中的功效。(图9)。给小鼠接种TC-1细胞7天后,在第0天,第7天,第14天将PBS,五肽,抗原肽及多肽疫苗皮下注射到小鼠体内(图9a)。在21天内监测不同处理组小鼠的肿瘤体积和体重(图9,b和c)。在监测期间,所有治疗小鼠的体重没有出现明显变化(图9c),表明多肽疫苗具有较好的生物相容性。用游离抗原肽和多肽疫苗治疗小鼠后,小鼠体内肿瘤的生长受到不同程度的抑制。与其他治疗组相比,用纳米纤维疫苗AmpF-E7治疗小鼠可显着抑制肿瘤生长,证明了纳米纤维疫苗在肿瘤免疫疗法中的优异功效。
通过流式细胞术分析T细胞向肿瘤组织的浸润,以研究抑制小鼠肿瘤生长的潜在机制(图9e)。当用PBS和五肽AmpF或PF治疗小鼠时,其CD4+或CD8+T细胞的浸润率非常低,而用多肽疫苗注射的小鼠解剖出的肿瘤组织中所含CD4+或CD8+T细胞的浸润率超过2%。另外,与纳米颗粒疫苗相比,纳米纤维疫苗AmpF-E7治疗组导致CD4+或CD8+T细胞向肿瘤组织的浸润率增强。用多肽疫苗AmpF-E744-57治疗的小鼠的肿瘤组织中CD4+或CD8+T细胞的浸润率分别为5.39%和6.07%。这些结果表明活化的T细胞浸润到肿瘤组织中,从而通过免疫治疗机制裂解肿瘤细胞。
还分析了用PBS,五肽,抗原肽及多肽疫苗治疗的小鼠脾脏中T淋巴细胞的增殖和免疫细胞因子的分泌,以确认抑制肿瘤的免疫治疗机制(图10)。在用纳米纤维肽疫苗AmpF-E749-57治疗小鼠后,脾脏中CD8+T细胞的增殖能力很强,在多肽疫苗AmpF-E744-57治疗组的小鼠身上解剖出来的脾脏中观察到了高含量的CD4+和CD8+T细胞。(图10,a和b)。另外,用纳米纤维和纳米颗粒肽疫苗接种的小鼠的脾脏中可检测到CD4+和CD8+T细胞分泌的INF-γ(图10,d和e)。然而,用纳米纤维疫苗治疗的小鼠脾脏中INF-γ明显高于纳米粒子疫苗治疗组。这些结果表明,与纳米颗粒多肽疫苗相比,纳米纤维多肽疫苗AmpF-E7诱导了脾脏T淋巴细胞的快速增殖和分化,这可能归因于有效的淋巴引流和延长的滞留时间。
为了研究多肽疫苗治疗期间T淋巴细胞在免疫反应中的作用,进一步估计了小鼠血清中免疫促进细胞因子IL-2和TNF-α的水平(图10,f和g)。流式细胞仪分析表明,与其他治疗组相比,纳米纤维疫苗AmpF-E7治疗组的小鼠血液中的这两种免疫促进细胞因子含量更高。为了验证多肽疫苗能够引起免疫记忆反应的激活,还评估了不同治疗组的小鼠的LNs中中央记忆T(TCM)细胞的增殖(图10c)。流式细胞仪结果显示用纳米纤维疫苗AmpF-E7治疗的小鼠中检测到的TCM细胞(CD44HiCD62L+)的含量比PF-E7多肽疫苗高出近1.7倍。这些结果表明纳米纤维疫苗可促进小鼠免疫细胞因子的分泌和免疫记忆的产生,从而成为针对HPV相关肿瘤的有效的自佐肽疫苗。
为了评估多肽疫苗的组织病理学毒性,收集不同治疗组的小鼠的肿瘤组织进行了常规苏木精和伊红(H&E)染色(图10h)。不同治疗组的小鼠的肿瘤组织染色结果显示了不同水平的细胞凋亡,其中纳米纤维疫苗AmpF-E7处理诱导了最显著的细胞死亡,证实了多肽疫苗AmpF-E7的增强的免疫治疗功效。还进行了免疫组织化学染色测定,以进一步验证不同治疗组的小鼠中T淋巴细胞的肿瘤浸润情况(图10,i和j)。抗CD8+免疫组织化学分析表明,两种纳米纤维疫苗治疗组的小鼠均导致CD8+T淋巴细胞在肿瘤细胞周围大量富集。然而,在抗CD4+免疫组织化学测定中,多肽疫苗AmpF-E744-57治疗组观察到CD4+T淋巴细胞在肿瘤组织附近的大量富集,因此,免疫组织化学染色测定法证明了纳米纤维疫苗诱导的细胞毒性T淋巴细胞向肿瘤组织的浸润增强,从而促进肿瘤细胞死亡并抑制肿瘤生长。
多肽疫苗与抗PD-1抗体的联合免疫疗法
进一步研究了多肽疫苗结合抗PD-1抗体的组联合免疫疗法针对HPV相关TC-1肿瘤的治疗功效(图11)。在第0天,第7天,第14天给TC-1肿瘤小鼠皮下注射PBS,抗PD-1,抗原肽E744-57+抗PD-1,多肽疫苗PF-E744-57+抗PD-1和多肽疫苗AmpF-E744-57+抗PD-1(图11a)。抗PD-1,抗原肽E744-57+抗PD-1和多肽疫苗PF-E744-57+抗PD-1治疗组均可不同程度的抑制肿瘤生长,但是多肽疫苗AmpF-E744-57+抗PD-1治疗组可以显著抑制小鼠肿瘤的生长(图11b)。所有治疗组小鼠的恒定体重表明多肽疫苗的系统毒性可忽略不计(图11d)。还通过流式细胞术分析了不同治疗组的小鼠T淋巴细胞的肿瘤浸润情况(图11e)。与抗PD-1,抗原肽E744-57+抗PD-1,多肽疫苗PF-E744-57+抗PD-1相比,多肽疫苗AmpF-E744-57+抗PD-1的组合显著增强了CD4+和CD8+T细胞的肿瘤浸润(图11,f和g),其中肿瘤中CD4+和CD8+T细胞的含量约为12%和9.1%。因此,的结果证明了多肽疫苗和抗PD-1抗体的联合使用对肿瘤免疫治疗具有协同作用,从而进一步提高纳米纤维多肽疫苗免疫治疗效果。
另外,利用ELISA试剂盒测定了不同治疗组小鼠血液中免疫细胞因子IFN-γ,IL-2和TNF-α的含量(图11,h-j)。结果表明,多肽疫苗AmpF-E744-57和抗PD-1联合治疗可促进IFN-γ,IL-2和TNF-α的产生,含量均高于任何其他治疗组。
通过H&E和免疫组化染色测定,评估不同治疗组小鼠肿瘤组织的细胞凋亡和T淋巴细胞浸润情况(图11,k-m)。不同治疗组的小鼠的肿瘤组织的H&E染色图像显示了不同水平的细胞凋亡。多肽疫苗AmpF-E744-57+抗PD-1联合治疗组的小鼠的肿瘤组织观察到明显的细胞死亡,证实了多肽疫苗AmpF-E744-57+抗PD-1联合治疗的增强的免疫治疗功效。免疫组织化学染色图像显示,抗PD-1,抗原肽E744-57+抗PD-1和多肽疫苗PF-E744-57+抗PD-1治疗组引起部分T淋巴细胞的浸润,从而导致部分肿瘤细胞的凋亡。然而,多肽疫苗AmpF-E744-57和抗PD-1的组合免疫疗法显着增强了CD4+和CD8+T淋巴细胞对肿瘤组织的浸润,进而诱导了强烈的免疫反应并导致肿瘤细胞大规模凋亡。这些结果表明,多肽疫苗AmpF-E744-57和抗PD-1的组合免疫疗法可促进T淋巴细胞的浸润和多种免疫促进细胞因子的产生,从而引发有效的肿瘤免疫反应。
Claims (3)
1.一种形貌可控自佐剂宫颈癌多肽疫苗的制备方法,其特征是:该形貌可控自佐剂宫颈癌多肽疫苗为纳米纤维多肽疫苗AmpF-E744-57,所述制备方法包括在制备多肽疫苗之前,将多肽冻干粉AmpF、AmpFE744-57溶解在水中来制备浓度为5mM的不同多肽的储备溶液的步骤;所述纳米纤维多肽疫苗AmpF-E744-57是通过将多肽AmpF和AmpFE744-57的溶液以90:10的摩尔比混合制备的,最终的总浓度为2mM;
其中所述AmpF为:
其中:R1为-NH2;R2为-OH;
AmpFE744-57为:
其中:R1为-NH2;R2为E744-57;
所述E744-57为:
E744-57的氨基酸序列为QAEPDRAHYNIVTF。
2.根据权利要求1所述的形貌可控自佐剂宫颈癌多肽疫苗的制备方法,其特征是:多肽AmpF、AmpFE744-57是通过标准Fmoc固相肽合成方法合成的。
3.一种权利要求1或2所述的形貌可控自佐剂宫颈癌多肽疫苗的制备方法得到的多肽疫苗。
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