CN114569711B - 一种预防弓形虫病的ME49Δcdpk3减毒活疫苗及其制备方法和应用 - Google Patents
一种预防弓形虫病的ME49Δcdpk3减毒活疫苗及其制备方法和应用 Download PDFInfo
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- CN114569711B CN114569711B CN202210298777.5A CN202210298777A CN114569711B CN 114569711 B CN114569711 B CN 114569711B CN 202210298777 A CN202210298777 A CN 202210298777A CN 114569711 B CN114569711 B CN 114569711B
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Abstract
本发明涉及弓形虫疫苗技术领域,具体来说是一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗,减毒活疫苗是通过CRISPR/Cas9介导的基因编辑技术在ME49上敲除CDPK3构建的ME49Δcdpk3虫株,减毒活疫的制备步骤包括(1)构建靶向Cas9质粒、(2)构建同源供体DNA、(3)构建ME49Δcdpk3的混合克隆虫株和(4)筛选获得ME49Δcdpk3单克隆虫株。在本申请中使用ME49Δcdpk3的速殖子免疫BALB/c小鼠,通过小鼠模型评估ME49Δcdpk3减毒株作为疫苗的保护性免疫,研究发现ME49Δcdpk3免疫引起的免疫反应可以保护小鼠免受急性和潜伏弓形虫感染,表明ME49Δcdpk3的速殖子可以提供对不同寄生虫菌株的有效保护,因此ME49Δcdpk3可被用作对抗弓形虫的潜在候选疫苗。
Description
技术领域
本发明涉及弓形虫疫苗技术领域,具体来说是一种预防弓形虫病的ME49Δcdpk3减毒活疫苗及其制备方法和应用。
背景技术
刚地弓形虫(T.gondii)是一种常见的人畜共患细胞内寄生虫,能够感染包括人类在内的几乎所有温血动物。世界上大约30%的人口感染了刚地弓形虫,在健康个体中大多数是无症状的,但在免疫功能低下或缺陷的人群可导致免疫缺陷病人出现弓形虫脑炎、视网膜脉络膜炎,母体妊娠期感染可引起先天性弓形虫病。此外,山羊等许多重要家畜的初次感染会导致流产和死胎,给畜牧业带来重大经济损失和严峻挑战。在动物或人类中,针对速殖子的乙胺嘧啶和磺胺嘧啶的固定组合是目前治疗急性期弓形虫病的首选药物,但此药物已经被证明对缓殖子或潜伏期感染无效。
弓形虫的生命周期复杂,传播途径多。家猫和其他野猫是刚地弓形虫的最终宿主,摄入猫***的卵囊是中间宿主(人和动物)感染的主要来源。此外,弓形虫还可以通过无性繁殖和捕食在中间宿主之间传播。弓形虫菌株具有复杂的种群结构。在北美和欧洲存在三种主要类型的弓形虫菌株(I、II和III)。Ⅰ型毒株以RH和GT1为主,Ⅱ型毒株PRU、ME49,Ⅲ型毒株以CEP为主,毒力差异较大。与北美和欧洲的弓形虫菌株相比,南美洲的弓形虫菌株在基因上更加多样化。然而,基因型Chinese 1已被确定为东亚的主要菌株,特别是在中国。Jensen等人发现来自不同基因型的菌株可以反复感染同一宿主。以上几种原因对弓形虫病的控制提出了很大的挑战。因此,开发有效的弓形虫病疫苗对于限制各种弓形虫菌株的感染至关重要。
近年来,相关研究人员已经进行了多种开发安全有效的弓形虫疫苗的研究。已开发出核酸疫苗、重组蛋白疫苗和鸡尾酒抗原疫苗来预防弓形虫感染。然而,这些疫苗都难以提供足够的保护。到目前为止,获得有效弓形虫疫苗最有希望的策略是使用减毒活疫苗,它可以诱导更高和更长期的保护性细胞和体液免疫反应来预防弓形虫感染。目前,是弓形虫的减毒活S48弓形虫株,是唯一商业化疫苗用于预防绵羊和山羊先天性弓形虫感染,研究出更多的弓形虫弱毒疫苗以扩大其应用范围,对于人类健康和畜牧养殖业的发展均有重要价值和意义。
刚地弓形虫拥有14个编码Ca2+依赖性蛋白激酶(CDPK)的基因,这些基因参与弓形虫的运动、入侵、增殖和逸出等多种功能。TgCDPK3对于快速诱导寄生虫排出和在小鼠体内建立慢性感染至关重要,它是体内寄生虫毒力的关键。在本申请中使用ME49Δcdpk3的速殖子免疫BALB/c小鼠,通过小鼠模型评估ME49Δcdpk3减毒株作为疫苗的保护性免疫效果。研究发现ME49Δcdpk3免疫引起了强烈的免疫反应并保护小鼠免受急性和潜伏弓形虫感染,表明ME49Δcdpk3的速殖子可以提供对不同寄生虫菌株的有效保护,因此ME49Δcdpk3可被用作对抗弓形虫的潜在候选疫苗。
发明内容
本发明所要解决的技术问题在于如何制备一种预防弓形虫病的ME49Δcdpk3减毒活疫苗,所制备的减毒活疫苗能够有效抵抗弓形虫的急性感染和慢性感染。
本发明通过以下技术手段实现解决上述技术问题的:
本发明第一方面提供一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗,所述减毒活疫苗是通过CRISPR/Cas9介导的基因编辑技术在ME49虫株敲除CDPK3构建ME49Δcdpk3虫株。
本发明第二方面提供一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗的制备方法,包括以下具体步骤:
(1)构建靶向Cas9质粒
敲除弓形虫CRISPR/Cas9的质粒pSAG1::Cas9-U6::sgUPRT,使用E-CRISPR软件进行靶点设计,使用定点突变试剂盒将质粒pSAG1::Cas9-U6::sgUPRT中的sgRNA替换为靶向CDPK3的sgRNA,即构建质粒pSAG1::Cas9-U6::sgCDPK3;
(2)构建同源供体DNA
将从II型ME49菌株的基因组DNA中扩增CDPK3 5'-和3'-同源臂片段,以及从pUPRT-DHFR质粒中扩增DHFR-TS序列;使用多片段一步快速克隆试剂盒将三片段通过同源重组克隆到pUC19线性化载体之间,构建三片段的供体DNA质粒;
(3)构建ME49Δcdpk3的虫株
将步骤(1)构建的质粒pSAG1::Cas9-U6::sgCDPK3和步骤(2)构建的供体DNA片段电穿孔转染到纯化的ME49速殖子中,并用乙胺嘧啶筛选,制备得到ME49Δcdpk3速殖子的混合克隆;
(4)筛选ME49Δcdpk3单克隆虫株
通过倍比稀释将单个弓形虫接种到铺有人***成纤维细胞(HFF细胞)的96孔板中;7-10天后通过PCR和免疫印迹检测阳性克隆,获得单个克隆的虫株。
优选地,所述步骤(1)中质粒pSAG1::Cas9-U6::sgUPRT的引物为:
CDPK3-gRNA-F:TGTACCGAGGGTTTTAGAGCTAGAAATAGC;
CDPK3-gRNA-R:CCTCCATGACAACTTGACATCCCCATTTAC。
优选地,所述步骤(2)中DHFR-TS的引物为:
DHFR-TS-F:TGTCATTCGATTTTCACCCCC;
DHFR-TS-R:AGTGTGATGACTCCGCAACT
GGATCGATCCCCCCGGGCTGC。
优选地,所述步骤(4)中诊断性PCR包括PCR1、PCR2和PCR3。
所述PCR1的引物为:
PCR1-F:GCCTAACAAGGATTCGATCAGTAGC;
PCR1-R:TGTCGTGGATTTACCAGTCATGGAC;
所述PCR2的引物为:
PCR2-F:TGACTCTTCATGTGGCATTTCACAC;
PCR2-R:TACTGTGTTAGGTAGCAAATGTGG;
所述PCR3的引物为:
PCR3-F:TCGTGCGTCTTCAGGCATGTACATC;
PCR3-R:GAGGTCCTTTCGCTTCCTGAGACTC。
优选地,所述ME49Δcdpk3速殖子的免疫剂量103~106个。
优选地,所述ME49Δcdpk3速殖子的免疫剂量为103个。
本发明第三方面提供一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗在预防寄生虫感染中的应用。
本发明第四方面提供一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗在预防弓形虫慢性感染中的应用。
本发明第五方面提供一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗在预防多种弓形虫急性感染中的应用。
本发明的优点在于:
1.本申请接种ME49Δcdpk3速殖子的小鼠可有效抵御多种野生型菌株,包括I型RH、II型ME49、Chinese1 WH3和WH6感染,表明ME49Δcdpk3疫苗接种在受到不同菌株的攻击时会引发强烈的免疫反应抵抗弓形虫的感染,而且ME49Δcdpk3免疫小鼠的再次感染弓形虫时腹膜液中的寄生虫负荷也显着降低。
2.本申请103个ME49Δcdpk3速殖子感染的小鼠临床评分显示小鼠的状态相对较好,且103个ME49Δcdpk3速殖子感染的小鼠血清中诱导了弓形虫特异性IgG具有相似的高水平,表明103个ME49Δcdpk3速殖子疫苗接种是一种安全有效的免疫剂量。
3.本申请小鼠血清中弓形虫IgG水平在整个疫苗接种期间保持高水平且稳定;对于IgG亚类水平,免疫小鼠中IgG1和IgG2a的水平较未免疫小鼠显着升高,并且IgG2a水平明显高于IgG1表明ME49Δcdpk3疫苗接种触发了以Th1反应为主的Th1和Th2混合免疫反应,说明ME49Δcdpk3疫苗接种存在潜在保护性免疫。
4.本申请接种疫苗75天的小鼠脾细胞上清中IFN-γ、TNF-α、IL-12p70和IL-10水平较未接种疫苗的小鼠显着升高,表明ME49Δcdpk3疫苗接种有效地引起细胞介导的免疫应答。
5.本申请的ME49Δcdpk3还减弱了小鼠慢性弓形虫病的发展。与未接种疫苗的小鼠相比,接种疫苗的小鼠感染50个ME49包囊后存活率为100%,在感染包囊35天后存活小鼠脑内包囊明显减少。
6.本申请接种ME49Δcdpk3疫苗的小鼠血清可以减少寄生虫的增殖,表明ME49Δcdpk3疫苗接种小鼠血清可以有效的抵抗弓形虫感染。
总之,本申请的ME49Δcdpk3疫苗接种可引发细胞免疫和体液免疫,并保护小鼠免受弓形虫感染,表明ME49Δcdpk3菌株可能是针对急性和潜伏弓形虫病的可行减毒活疫苗候选者。
附图说明
图1为本申请实施例2中PCR和免疫印迹检测结果。
图2为本申请实施例9中寄生虫感染期间小鼠的临床症状和小鼠血清中的弓形虫特异性IgG的水平检测结果。
图3为本申请实施例10中ME49Δcdpk3疫苗免疫保护小鼠免受弓形虫速殖子感染情况。
图4为本申请实施例11中ME49Δcdpk3疫苗接种保护小鼠免受包囊感染情况。
图5为本申请实施例12中ME49Δcdpk3免疫后30天、75天和125天小鼠血清中的细胞因子水平。
图6为本申请实施例12中ME49Δcdpk3免疫后30天、75天和125天小鼠血清中弓形虫特异性总IgG和IgG亚类(IgG1和IgG2a)水平。
图7为本申请实施例13中弓形虫抗原刺激后ME49Δcdpk3疫苗接种诱导的细胞免疫反应结果。
图8为本申请实施例14中使用ME49Δcdpk3疫苗接种小鼠血清进行被动免疫结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
本实施例公开一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗,减毒活疫苗是通过CRISPR/Cas9介导的基因编辑技术在ME49虫株敲除CDPK3构建的ME49Δcdpk3虫株。
实施例2
本实施例公开一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗的制备方法,包括以下具体步骤:
(1)构建靶向Cas9质粒
敲除弓形虫CRISPR/Cas9的质粒pSAG1::Cas9-U6::sgUPRT(购自美国Addgene,型号为#54467)。使用E-CRISPR软件进行靶点设计,并使用定点突变试剂盒(购自NewEngland Biolabs(NEB)公司)将质粒pSAG1::Cas9-U6::sgUPRT中的sgRNA替换为靶向CDPK3的sgRNA,即构建质粒pSAG1::Cas9-U6::sgCDPK3。
其中,质粒pSAG1::Cas9-U6::sgUPRT的引物为:
CDPK3-gRNA-F:TGTACCGAGGGTTTTAGAGCTAGAAATAGC;
CDPK3-gRNA-R:CCTCCATGACAACTTGACATCCCCATTTAC。
(2)构建同源供体DNA
将从II型ME49菌株的基因组DNA中扩增CDPK3 5'-和3'-同源臂片段,以及从pUPRT-DHFR中扩增DHFR-TS序列。使用多片段一步快速克隆试剂盒(ClonExpress MultiSOne Step Cloning Kit,购自中国南京的Vazyme Biotech公司)将CDPK3 5'-同源臂片段、CDPK3 3'-同源臂片段以及DHFR-TS序列这三个片段通过同源重组克隆到pUC19线性化载体之间,构建三片段的供体DNA质粒。
其中,CDPK3 5'同源臂片段UpCDPK3-F的引物为:
AAAACGACGGCCAGTGAATTC
AGCCAACATGCATTGGAGCT;
UpCDPK3-R的引物为:
GGGGGTGAAAATCGAATGACA
ACGCAGCGACTGGGAGAATC。
步骤(2)中DHFR-TS的引物为:
DHFR-TS-F:TGTCATTCGATTTTCACCCCC;
DHFR-TS-R:AGTGTGATGACTCCGCAACT
GGATCGATCCCCCCGGGCTGC。
CDPK3 3'同源臂片段DnCDPK3-F的引物为:
GCAGCCCGGGGGGATCGATCC
AGTTGCGGAGTCATCACACT;
DnCDPK3-R的引物为:
GACCATGATTACGCCAAGCTT
TCGTTGTGGGCTATACAGCT。
(3)构建ME49Δcdpk3的虫株
将步骤(1)构建的质粒pSAG1::Cas9-U6::sgCDPK3和步骤(2)构建的供体DNA片段电穿孔转染到纯化的ME49速殖子中,并用1μM的乙胺嘧啶筛选,制备得到ME49Δcdpk3速殖子的混合克隆。
(4)筛选获得ME49Δcdpk3单克隆虫株
通过倍比稀释将单个弓形虫接种到铺有人***成纤维细胞(HFF细胞)的96孔板中;7-10天后通过PCR和免疫印迹检测阳性克隆,获得单个克隆的虫株,即为ME49Δcdpk3虫株。
其中,诊断性PCR包括PCR1、PCR2和PCR3。
PCR1的引物为:
PCR1-F:GCCTAACAAGGATTCGATCAGTAGC;
PCR1-R:TGTCGTGGATTTACCAGTCATGGAC;
PCR2的引物为:
PCR2-F:TGACTCTTCATGTGGCATTTCACAC;
PCR2-R:TACTGTGTTAGGTAGCAAATGTGG;
PCR3的引物为:
PCR3-F:TCGTGCGTCTTCAGGCATGTACATC;
PCR3-R:GAGGTCCTTTCGCTTCCTGAGACTC。
ME49Δcdpk3菌株的PCR鉴定结果如图1A,并以ME49野生型(WT)菌株用作对照,PCR1和PCR2分别验证5'和3'同源臂的整合,而PCR3验证CDPK3基因的成功敲除,可以看出CDPK3基因被成功敲除,获得ME49敲除CDPK3的单克隆虫株。
ME49 WT和ME49Δcdpk3虫株蛋白质印迹检测结果如图1B,以弓形虫肌动蛋白(actin)抗体用作内参,印迹结果显示ME49Δcdpk3菌株不表达CDPK3蛋白。
实施例3
本实施例利用实施例2制备的ME49Δcdpk3速殖子对小鼠进行寄生虫感染试验,以评估CDPK3失活对寄生虫毒力的影响。
为了测试ME49和ME49Δcdpk3速殖子感染小鼠之间免疫反应的差异,分别用ME49和ME49Δcdpk3寄生虫感染小鼠,使用剂量为103、104、105和106个,并观察小鼠35天内的临床症状。
临床评分范围从0(无体征)到10(所有体征)。评估的临床症状包括驼背、立毛、行为、寻虫、上睑下垂、眼睛凹陷、共济失调、动作缓慢、疏散和触觉反射差以及趴着。此外,在感染后第35天收集上述感染小鼠的血清,并通过10μg/ml可溶性速殖子抗原包被的ELISA板测定弓形虫特异性IgG水平。
实施例4
本实施例利用实施例2制备的ME49Δcdpk3速殖子对小鼠进行急性和慢性弓形虫感染试验。
对BALB/c小鼠通过腹腔注射103个ME49Δcdpk3速殖子进行免疫。在免疫后第75天,分别对不同的小鼠注射103个Chinese1 WH3(每组10只小鼠)、105个Chinese1 WH6(每组10只小鼠)或50个ME49包囊(每组10只小鼠)。在免疫后第125天,给小鼠注射103个RH和105个ME49速殖子(每组10只小鼠)。用相同剂量和途径感染的未接种疫苗的小鼠作为对照。然后每天监测再次感染的小鼠35天内的临床症状和存活率。在弓形虫感染后的第7天,确定小鼠的腹膜液寄生虫负荷。对于慢性感染,在攻击后第35天检测到存活小鼠大脑中的包囊数量。
实施例5
本实施例利用实施例2制备的ME49Δcdpk3速殖子对小鼠进行免疫,然后检测免疫小鼠血清中细胞因子和弓形虫特异性IgG水平。
对BALB/c小鼠通过腹腔注射103个ME49Δcdpk3速殖子进行免疫,并用300μl的PBS腹腔注射模拟接种,分别在接种后30天、75天和125天获得小鼠血清样本。并通过酶联免疫吸附试验(ELISA)检测血清总弓形虫特异性IgG和IgG亚型(IgG1和IgG2a)的水平。
具体操作为:将在包被缓冲液(50mM碳酸盐缓冲液,pH9.6)中稀释的10μg/ml可溶性弓形虫抗原用于包被96孔ELISA板,然后将ELISA板在4℃下孵育过夜,然后用含有0.05%Tween-20(PBST,PH7.4)的磷酸盐缓冲盐水洗涤5次;再使用3%的BSA在37℃下阻断非特异性结合1小时,然后用PBST洗涤五次。
然后,将收集的血清按1:50稀释后,在37℃下孵育1小时;洗涤后,将HRP螯合的山羊抗小鼠IgG和和IgG亚型(IgG1或IgG2a)用PBST(1:1000)稀释然后添加到96孔板中,每孔100μl,并在37℃孵育一小时;然后每个孔用PBST洗涤5次。
最后,使用TMB(100μl/孔)作为底物进行反应,加入2M的H2SO4以停止反应。用ELISA读数器在450nm处测量光密度(OD),一式三份,以此分析所有血清样品。同时,根据试剂盒说明书的建议,使用ELISA试剂盒测量细胞因子IFN-γ、IL-12p70、TNF-α和IL-10的产生水平。
实施例6
本实施例对实施例5中免疫后75天分离来自接种和未接种小鼠的脾细胞上清液中细胞因子的产生水平进行评估。
将免疫和未免疫的小鼠麻醉并处死,采集脾脏以评估脾细胞细胞因子产生的水平。具体操作为:脾细胞通过70μM金属丝网筛分离,并在裂解缓冲液中溶血5分钟以获得单个脾细胞悬液;脾细胞在24孔板中培养,并用ME49菌株的10μg/mL弓形虫可溶性速殖子抗原(STAg)刺激;然后收集无细胞上清液,通过ELISA测量培养后72小时的TNF-α和白细胞介素10(IL-10)的水平、培养后96小时的白细胞介素12p70(IL-12p70)和干扰素γ(IFN-γ)的水平,以未免疫的小鼠脾细胞用作阴性对照。
实施例7
本实施例利用实施例5中接种ME49Δcdpk3速殖子的小鼠进行被动免疫。
对BALB/c小鼠腹腔注射106个II型ME49速殖子,在攻击后的0天和3天,将来自125天的正常小鼠或免疫小鼠的血清通过尾静脉注射到受感染的小鼠中(100μl/小鼠),正常小鼠的血清用作阴性对照。在攻击后7天通过荧光定量PCR检测腹膜液中的寄生虫负荷,以测量被动免疫下的寄生虫增殖,并每天记录每组剩余小鼠35天的死亡情况。
实施例8
本实施例对受感染小鼠中寄生虫负荷进行测定。
使用Steady Pure Genomic DNA Kit从寄生虫感染小鼠的腹膜液中提取基因组DNA,使用相应的基因组DNA样本,通过qPCR扩增Tg-529基因(正向引物为5'-CGTCCAGGGAGGAAGACGAAAGTTG-3',反向引物为5'-CGCTGCAGACAGAGTGCATCTGGATT-3')确定寄生虫负荷。每个样品中的小鼠肌动蛋白基因用作内参(正向引物为5'-AGCTTCTTTGCAGCTCCTTCGT-3',反向引物为5'-TACACGCTAGGCGTAAAGTTGG-3')。根据说明书,使用Premix Ex TaqTM II试剂盒进行实时qPCR,所有的反应在Roche LC480II***上运行。
获得用于定量寄生虫的标准曲线:提取来自0、100、101、102、103、104、105、106和107种寄生虫的基因组DNA,然后使用基于Tg529的qPCR获得每个样品的CT值作为纵坐标,Lg(速殖子数)作为横坐标,并根据CT值和Lg生成标准曲线,再根据标准计算寄生虫负荷。
实施例9
对实施例3中小鼠感染ME49和ME49Δcdpk3后35天的临床症状进行监测,并未感染的小鼠用作对照,监测结果如图2A。结果表明,与野生型(WT)菌株ME49相比,ME49Δcdpk3速殖子的毒力明显减弱,即使在1×106速殖子的感染剂量下,存活率仍为100%。
对实施例3中103、104、105和106个亲代菌株ME49或ME49Δcdpk3速殖子感染的BALB/c小鼠进行感染后35天的临床评估结果如图2B。初步实验结果表明,在103、104、105和106个ME49Δcdpk3速殖子感染的小鼠观察到较低的临床评分,而103、104、105和106个ME49感染的小鼠表现出较严重的临床症状。
对实施例3中103、104、105和106个亲代菌株ME49或ME49Δcdpk3速殖子感染的BALB/c小鼠进行感染后35d的IgG进行检测,并以未感染的血清作为对照,结果如图2C(统计差异****p<0.0001,每组6只小鼠)。结果表明,在感染后35天,在103、104、105和106个ME49Δcdpk3速殖子或103、104、105个ME49感染的小鼠血清中诱导了弓形虫特异性IgG具有相似的高水平。这些代表了相似的免疫原性,结合临床评分表明103个ME49Δcdpk3速殖子疫苗接种是一种安全有效的免疫剂量。
实施例10
以实施例4中接种后75天用103个Chinese1 WH3或105个Chinese1 WH6感染接种疫苗小鼠的死亡率,评估ME49Δcdpk3免疫对速殖子感染的保护功效,并以未免疫的小鼠被认为是阴性对照,结果如图3A-B。可以看出,感染Chinese 1WH6的小鼠的存活率为100%。然而,当用强毒性Chinese1 WH3攻击时,尽管小鼠的存活时间已显着延长,但只有30%的接种小鼠存活。
以实施例4中接种后125天用103个I型(RH)或105个II型(ME49)感染接种疫苗小鼠的死亡率,检查ME49Δcdpk3疫苗接种是否提供长期保护性免疫,并以未免疫的小鼠被认为是阴性对照,结果如图3D-E。可以看出,用ME49速殖子攻击的免疫小鼠中没有观察到死亡,而用RH速殖子再次攻击的接种小鼠的存活率约为30%,表明ME49Δcdpk3疫苗接种对毒性较小的寄生虫菌株具有更持久和更强的保护作用,但对毒性较强菌株的保护作用较弱。
在实施例4中感染后7天收集的ME49Δcdpk3免疫小鼠二次感染腹膜液,以评估小鼠腹膜液寄生虫负荷,并以未接种疫苗的受攻击小鼠作为对照,采用实施例8的定量PCR技术对小鼠中寄生虫负荷进行测定,结果如图3C、3F(统计差异由***p<0.001、**p<0.01、*p<0.05、ns:无差异)。可以看出,在未免疫小鼠中,RH、ME49和Chinese1WH3和Chinese1 WH6感染导致寄生虫快速增殖,而在RH、ME49和Chinese1 WH3和Chinese1 WH6攻击的ME49Δcdpk3疫苗接种小鼠中检测到非常少的寄生虫,表明ME49Δcdpk3疫苗接种促进了感染寄生虫的快速消除,进而说明ME49Δcdpk3免疫接种可产生有效且安全的保护性免疫,以清除具有挑战性的寄生虫,从而使宿主得以存活。
实施例11
在实施例4中接种了103个ME49Δcdpk3速殖子的BALB/c小鼠免疫后75天,小鼠口服感染50个ME49包囊,记录小鼠的存活率,以评估ME49Δcdpk3免疫对慢性感染的保护功效,并以未接种疫苗的小鼠作为对照,结果如图4A。可以看出,接种ME49Δcdpk3疫苗的小鼠存活率为100%,而未接种疫苗的小鼠只有50%存活。
在感染包囊后35天,评估存活小鼠大脑中的寄生虫包囊负荷,存活的小鼠包括免疫和未免疫的,结果如图4B(统计差异***P<0.001,每组5只小鼠)。可以看出,未接种疫苗的每个小鼠大脑的包囊数量为1640±638.7个,而接种ME49Δcdpk3的每个小鼠大脑的包囊数量为94.00±21.04(p<0.001),结果表明ME49Δcdpk3菌株还诱导针对缓殖子感染的保护性免疫反应。
实施例12
通过实施例5中BALB/c小鼠接种ME49Δcdpk3速殖子后30天、75天和125天的小鼠血清评估IL-12p70、IL-10、IFN-γ和TNF-α水平,来阐明ME49Δcdpk3疫苗接种提供的免疫反应机制,并以未免疫小鼠的血清样品作为阴性对照,结果如图5(统计差异由***p<0.001、**p<0.01、*p<0.05、ns:无差异,每组6只小鼠)。其中,A对应IFN-γ、B对应IL-12p70、C对应TNF-α、D对应IL-10。可以看出,与未免疫小鼠相比,促炎细胞因子IL-12p70、IFN-γ和TNF-α水平在免疫后30天、75天和125天时显着升高。然而,75天的细胞因子水平低于30天的细胞因子水平,这可能是由于抗炎反应的激活,感染后30天接种疫苗的小鼠IL-10水平升高就证明了这一点。在125天时,接种小鼠的细胞因子水平与未接种小鼠的细胞因子水平相当。
实施例5中小鼠血清样本弓形虫特异性IgG和IgG亚类水平,并以未免疫小鼠的血清样品作为阴性对照,测量结果如图6(统计差异由***p<0.001、**p<0.01、*p<0.05、ns:无差别,每组6只小鼠)。评估弓形虫特异性IgG水平,观察到ME49Δcdpk3免疫后的第30天、第75天和第125天小鼠IgG水平的增加。通过检测IgG亚类(IgG1和IgG2a)的水平,确定ME49Δcdpk3触发的免疫反应类型。结果表明,接种ME49Δcdpk3的小鼠在感染后第30天、第75天和第125天诱导高水平的IgG1和IgG2a抗体,并且IgG2a滴度明显高于IgG1,这表明ME49Δcdpk3诱导了偏向Th1的免疫反应。以上结果表明,ME49Δcdpk3免疫还可以激活细胞和体液免疫反应以控制弓形虫感染。
实施例13
在实施例6中小鼠接种ME49Δcdpk3免疫后75天,分离来自接种和未接种小鼠的脾细胞,以评估ME49Δcdpk3接种动物中细胞介导的免疫反应。脾细胞在体外培养,并用刚地弓形虫速殖子可溶性抗原刺激,收集上清液并通过ELISA估计细胞因子水平,结果如图7(每组6只小鼠)。可以看出,与从未接种疫苗的小鼠获得的脾细胞相比,弓形虫可溶性抗原诱导了高水平的IFN-γ、TNF-α和IL-12p70以及IL-10,这些结果表明ME49Δcdpk3免疫可以激活有效的细胞免疫反应。
实施例14
实施例12已证明用ME49Δcdpk3免疫的小鼠具有高水平的弓形虫特异性IgG,为了确定该抗体在进一步限制寄生虫感染方面的作用,通过实施例7来确定免疫小鼠血清对弓形虫感染的保护性免疫结果如图8(统计差异*p<0.05)。其中,A为寄生虫负担,B为小鼠的存活率。可以看出,与接种正常血清的未免疫的小鼠相比,接种ME49Δcdpk3免疫小鼠血清的小鼠腹膜液寄生虫负荷显着降低,且免疫小鼠存活率明显高于未免疫鼠为60%。总之,这些结果表明接种ME49Δcdpk3的小鼠血清在一定程度上减少了寄生虫的增殖。
使用原理及优点:本申请使用103、104、105和106个亲代菌株ME49和ME49Δcdpk3弓形虫速殖子感染小鼠并监测小鼠的存活率,结果表明在1×106速殖子/小鼠的感染剂量下,ME49Δcdpk3弓形虫感染的小鼠在35天内的存活率仍为100%(图2A)。103个ME49Δcdpk3速殖子感染的小鼠显示出较轻的临床症状,并且103个ME49Δcdpk3速殖子感染的小鼠血清中诱导的弓形虫特异性IgG与103、104、105个ME49和104、105、106个ME49Δcdpk3速殖子感染的小鼠血清具有相似的高水平(图2B-C)。
为了获得减毒ME49Δcdpk3菌株所需的适当免疫原性而不在免疫小鼠中产生过度的免疫反应,本申请对每只小鼠施用103个ME49Δcdpk3速殖子。结果表明接种ME49Δcdpk3速殖子的小鼠可有效抵御多种野生型菌株,包括I型RH、II型ME49、Chinese1 WH3和WH6感染。表明ME49Δcdpk3疫苗接种在受到不同菌株的攻击时会引发免疫反应抵抗弓形虫的感染。值得注意的是,与毒性较低的II型ME49和Chinese1 WH6菌株(100%存活率)相比,对毒性较强的I型RH和Chinese1 WH3菌株的保护显着降低(30%存活率)(图3A,B,D,E)。此外,ME49Δcdpk3接种小鼠的腹膜液中的寄生虫负荷显着降低(p<0.001)(图3C,F)。
本申请的ME49Δcdpk3还减弱了小鼠慢性弓形虫病的发展。与未接种疫苗的小鼠相比,接种疫苗的小鼠感染50个ME49包囊后存活率为100%,脑内寄生虫包囊明显减少(图4)。
为了评估ME49Δcdpk3疫苗接种的潜在保护性免疫,本申请检测了小鼠血清中细胞因子和抗体的水平。弓形虫IgG水平在整个疫苗接种期间保持高水平且稳定(图6)。在测试IgG亚类时,发现与未免疫小鼠相比,免疫小鼠中IgG1和IgG2a的水平显着升高(以IgG2a为主)(图6)。这些表明ME49Δcdpk3疫苗接种触发了以Th1反应为主的Th1和Th2混合免疫反应。
先前的研究表明(如Romagnani S等(Annu Rev Immunol(1994)12:227-57.Epub1994/01/01.doi:10.1146/annurev.iy.12.040194.001303)、Liu等(Front Microbiol(2019)10:813.Epub 2019/05/21.doi:10.3389/fmicb.2019.00813.)、Dupont CD等(SeminImmunopathol(2012)34(6):793-813.Epub 2012/09/08.doi:10.1007/s00281-012-0339-3)),IFN-γ,IL12,TNF-α与Th1免疫反应相关,而IL-10与Th2反应相关,偏向Th1的免疫反应可有效预防发生的弓形虫感染。促炎细胞因子IFN-γ、IL-12和TNF-α对于激活细胞介导的针对弓形虫感染的免疫反应至关重要。IFN-γ可调节多种细胞内机制杀死寄生虫并抑制其复制,是抗弓形虫的主要介质。炎症因子IL-12可刺激CD4+和CD8+T细胞和自然杀伤细胞(NK)释放IFN-γ。TNF-α需要提供额外的信号和IFN-γ协同作用以杀死寄生虫。抗炎细胞因子IL-10负责在免疫反应的急性阶段下调IFN-γ。
接种疫苗后30天,与未接种疫苗的小鼠相比,接种疫苗的小鼠血清中IFN-γ、TNF-α、IL-12p70和IL-10水平显着升高,并在接种后125天恢复正常(图5),表明ME49Δcdpk3疫苗接种维持了细胞因子相对平衡水平。脾细胞刺激试验中高水平的促炎细胞因子IFN-γ、IL-12p70和TNF-α和抗炎细胞因子IL-10也证明了这种平衡(图7)。
本申请在小鼠接种ME49Δcdpk3疫苗后125天的血清通过尾静脉注射用于主动抵抗弓形虫的感染。试验表明,接种ME49Δcdpk3疫苗的小鼠血清可以减少寄生虫的增殖(图8)。
总之,本申请的ME49Δcdpk3疫苗接种可引发细胞免疫和体液免疫保护小鼠免受弓形虫感染,表明ME49Δcdpk3菌株可能是针对急性和潜伏弓形虫病的可行减毒活疫苗候选者。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗,其特征在于:所述减毒活疫苗是通过CRISPR/Cas9介导的基因组编辑技术在ME49虫株敲除CDPK3构建的ME49Δcdpk3虫株。
2.如权利要求1所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗的制备方法,其特征在于:包括以下具体步骤:
(1)构建靶向Cas9质粒
敲除弓形虫CRISPR/Cas9的质粒pSAG1::Cas9-U6::sgUPRT,使用E-CRISPR软件进行靶点设计,使用Q5定点突变试剂盒将质粒pSAG1::Cas9-U6::sgUPRT中的sgRNA替换为靶向CDPK3的sgRNA,即构建质粒pSAG1::Cas9-U6::sgCDPK3;所述质粒pSAG1::Cas9-U6::sgUPRT的引物为:
CDPK3-gRNA-F:TGTACCGAGGGTTTTAGAGCTAGAAATAGC;
CDPK3-gRNA-R:CCTCCATGACAACTTGACATCCCCATTTAC;
(2)构建同源供体DNA
将从II型ME49菌株的基因组DNA中扩增CDPK3 5'-和3'-同源臂片段,以及从pUPRT-DHFR中扩增DHFR-TS序列;使用多片段一步快速克隆试剂盒将三片段通过同源重组克隆到pUC19线性化载体之间,构建三片段的供体DNA质粒;DHFR-TS的引物为:
DHFR-TS-F:TGTCATTCGATTTTCACCCCC;
DHFR-TS-R:AGTGTGATGACTCCGCAACTGGATCGATCCCCCCGGGCTGC;
(3)构建ME49Δcdpk3的虫株
将步骤(1)构建的质粒pSAG1::Cas9-U6::sgCDPK3和步骤(2)构建的供体DNA片段电穿孔转染到纯化的ME49速殖子中,并用乙胺嘧啶筛选,制备得到ME49Δcdpk3的混合克隆;
(4)筛选获得ME49Δcdpk3单克隆虫株
通过倍比稀释将单个弓形虫接种到铺有人***成纤维细胞的96孔板中;7-10天后通过PCR和免疫印迹检测阳性克隆,获得单个克隆的虫株;PCR包括PCR1、PCR2和PCR3;
所述PCR1的引物为:
PCR1-F:GCCTAACAAGGATTCGATCAGTAGC;
PCR1-R:TGTCGTGGATTTACCAGTCATGGAC;
所述PCR2的引物为:
PCR2-F:TGACTCTTCATGTGGCATTTCACAC;
PCR2-R:TACTGTGTTAGGTAGCAAATGTGG;
所述PCR3的引物为:
PCR3-F:TCGTGCGTCTTCAGGCATGTACATC;
PCR3-R:GAGGTCCTTTCGCTTCCTGAGACTC。
3.根据权利要求1所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗,其特征在于:所述ME49Δcdpk3速殖子的免疫剂量103~106个。
4.根据权利要求3所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗,其特征在于:所述ME49Δcdpk3速殖子的免疫剂量为103个。
5.根据权利要求3所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗,其特征在于:所述ME49Δcdpk3速殖子的免疫剂量为105个。
6.如权利要求1所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗在制备预防刚地弓形虫感染的疫苗中的应用。
7.如权利要求1所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗在制备预防弓形虫慢性感染的疫苗中的应用。
8.如权利要求1所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗在制备预防弓形虫急性感染的疫苗中的应用。
9.如权利要求1所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗在制备引起宿主细胞免疫反应预防弓形虫感染的疫苗中的应用。
10.如权利要求1所述的一种用于预防弓形虫病的ME49Δcdpk3减毒活疫苗在制备引起宿主体液免疫反应预防弓形虫感染的疫苗中的应用。
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