弓形虫核苷三磷酸脱氢酶功能分析及疫苗候选靶标的研究
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摘要
刚地弓形虫(Toxoplasma gondii)是一种专性细胞内寄生的机会致病性原虫,在人和动物的有核细胞内寄生和繁殖,引起人兽共患的弓形虫病。尤其对于孕妇、婴儿和免疫低下者感染后危害极大。目前已成为发展中国家乃至全世界关注的机会感染性疾病之一。弓形虫在宿主体内快速增殖以及所产生的炎症反应可导致宿主机体出现严重的组织损伤和临床症状。虽然许多抗生素对急性弓形虫病治疗有效,但这些药物本身会对患者产生一定的副作用。因此,找到一种新药,特别是一种既能抑制弓形虫增殖又几乎不会产生副作用的药物成为弓形虫病治疗的当务之急。
弓形虫侵入宿主细胞后位于一个由纳虫泡膜包绕的纳虫泡内,位于纳虫泡内的虫体可通过分泌一些蛋白成分修饰纳虫泡内环境,从而维持自身生长发育需求。其中,核苷三磷酸脱氢酶(nucleoside triphosphate hydrolase, NTPase)是弓形虫适应宿主细胞内环境获得嘌呤的寄生机制而产生,对弓形虫在寄生、繁殖方面具有重要意义。由于弓形虫属于嘌呤营养缺陷型,无法通过从头途径合成嘌呤碱基,因此,为了生存和繁殖,弓形虫只能通过补救合成途径直接从宿主细胞获取嘌呤。弓形虫NTPase蛋白存在于速殖子体膜表面,但当速殖子侵入宿主细胞不久,NTPase即分泌入纳虫泡的网状管腔系统内,在二巯基化合物(如DTT)的激活作用下,能连续水解所有的三磷酸核苷和三磷酸脱氧核苷至单磷酸形式。感染宿主细胞的弓形虫速殖子即是利用NTPase分解宿主细胞来源的ATP,以合成维持自身生存所必需的嘌呤核苷酸。
弓形虫NTPase蛋白有NTPase-Ⅰ和NTPase-Ⅱ两种亚型。编码NTPase基因组DNA无内含子,由3个连续排列的开放读码框架(ORF)组成,分别为NTP1、NTP2和NTP3。其中NTP2不具备编码蛋白的能力,而NTPase-Ⅰ、NTPase-Ⅱ是分别与编码基因NTP3和NTP1相对应的一组同工酶。其中NTPase-Ⅰ仅存在于有毒株,而NTPase-Ⅱ几乎存在于所有弓形虫虫株。显而易见,以NTPase-Ⅱ蛋白作为药物或疫苗靶点将比NTPase-Ⅰ蛋白具有更广泛地应用前景。
本研究将以原核表达的NTPase-Ⅱ蛋白免疫BALB/c小鼠,制备特异性抗弓形虫核苷三磷酸水解酶(NTPase)Ⅱ型重组蛋白(rTgNTPase-Ⅱ)单克隆抗体,观察所获单克隆抗体的保护作用,阐明弓形虫NTPase-Ⅱ在RH株速殖子生长发育中的作用。同时观察rTgNTPase-Ⅱ免疫后,BALB/c小鼠体内所产生的免疫反应类型及免疫保护作用,为寻找新的疫苗靶点提供实验依据。
1.弓形虫NTPase-Ⅱ的基因克隆和蛋白表达
采用PCR方法扩增得到刚地弓形虫RH株NTPase基因,克隆入pGEM-T Easy载体,经酶切与测序鉴定后亚克隆至表达质粒pBAD-HisB,成功构建原核表达载体pBAD-HisB-NTPase,转入E.coli BL21(DE3)中获得高效表达,表达产物主要存在于包涵体中。融合蛋白的相对分子量(Mr)约70 kDa,与理论值相近。
使用Ni离子亲和层析柱纯化重组质粒pBAD-HisB-NTPase表达产生的含组氨酸的重组蛋白,SDS-PAGE鉴定纯度约为84%. Western blotting和ELISA分析结果显示,纯化的重组蛋白可被鼠抗重组蛋白血清特异性识别,具有良好的免疫反应性和免疫原性。
2.抗弓形虫rNTPase-Ⅱ单克隆抗体的制备及其功能分析
通过制备特异性抗弓形虫核苷三磷酸水解酶(NTPase)Ⅱ型重组蛋白(rTgNTPase-Ⅱ)单克隆抗体,观察所获单克隆抗体的保护作用,以此阐明弓形虫NTPase-Ⅱ在RH株速殖子生长发育中的作用。
将rTgNTPase-Ⅱ抗原(50μg/100μl)以福氏完全佐剂等体积混合后皮下多点注射免疫BALB/c小鼠,并以同样剂量的rTgNTPase-Ⅱ抗原等体积混合福氏不完全佐剂后每隔2周进行1次加强免疫,一共进行3次加强免疫。最后一次加强免疫后3天取免疫小鼠脾细胞与小鼠骨髓瘤Sp2/0细胞进行细胞融合,细胞融合率为92.71%(534/576)。利用纯化的rTgNTPase-Ⅱ作为包括抗原,以间接ELISA法进行筛选。对于筛选到的阳性克隆株以有限稀释法进行3次亚克隆,最终得到2株高亲和力杂交瘤细胞系,分别命名为MNT1与MNT2。将得到的单克隆抗体注射经降植烷处理的BALB/c小鼠腹腔,收集腹水以亲和层析法进行单抗纯化。
2.1单克隆抗体的鉴定：
采用Sigma抗体亚型检测试剂盒测定单克隆抗体的免疫球蛋白类别和亚类,证实2株单克隆抗体均为IgG2a亚类。间接ELISA法测定杂交瘤细胞株培养上清与小鼠腹水效价,检测结果表明,MNT1与MNT2细胞培养上清效价分别为1：12 800 and 1：6 400,而小鼠腹水效价分别为1：51200与1：12 800。同时将杂交瘤细胞株连续传代三个月及液氮冻存一个月后复苏,不同时间取其培养液上清,用间接ELISA法检测其抗体效价,测定抗体分泌的稳定性。结果发现,细胞连续传代三个月,分泌抗体的能力稳定。液氮冻存一个月后复苏,对细胞上清进行检测,抗体仍为阳性。以Western Blot法鉴定单克隆抗体的特异性,结果表明,两株单克隆抗体与rTgNTPase-Ⅱ蛋白在约70kDa处出现反应条带,与弓形虫RH株速殖子全虫抗原在约63kDa处出现反应条带,均与预期结果相符。而与阴性对照(含pBAD-HisB载体的DE3细菌裂解液)之间未出现反应条带。最后,将纯化的弓形虫RH株速殖子分别与单克隆抗体或阴性对照反应后,加入FITC标记的羊抗鼠IgG,利用间接荧光抗体实验(IFAT)于激光共聚焦显微镜下观察NTPase-Ⅱ在速殖子中的定位,发现2株单克隆抗体均与弓形虫RH株速殖子出现荧光反应。
2.2单克隆抗体保护性实验
2.2.1单克隆抗体抑制虫体入侵与胞内生长发育情况：将纯化的弓形虫RH株速殖子与单克隆抗体孵育后按照虫株数：细胞数为2：1的比例,对COS-7宿主细胞进行体外细胞培养。并于感染后3小时、9小时与15小时分别取出不同处理组于油镜下进行计数观察。其中与单抗孵育3h的细胞培养孔中观察100个宿主细胞中被速殖子感染的细胞数量(感染率),评价单抗对弓形虫RH株速殖子入侵宿主细胞的抑制作用。同时分别计数与单抗孵育3h、9h、15h的细胞培养孔中100个感染速殖子的阳性细胞中弓形虫虫体数量(感染度),评价单克隆抗体对弓形虫RH株在宿主细胞内生长发育的抑制作用。感染3小时后的感染率检测结果表明：MNT1、MNT2、IgG2a与完全培养液处理后的感染率分别为12%,10%,12%与13%,各组之间结果无统计学差异(χ2=0.458,P=0.928),表明2株单克隆抗体无抑制弓形虫RH株速殖子入侵COS-7宿主细胞的能力。然而在速殖子感染9h(χ2=19.741,P=0.000)与15h(χ2=44.974,P=0.000)后,各处理组间感染度均存在显著性差异,其中MNT1单克隆抗体处理组抑制虫体在COS-7细胞中增殖的效果最好,MNT2单克隆抗体处理组次之。表明2株单克隆抗体均可显著抑制弓形虫RH株速殖子在宿主细胞内的生长发育。且MNT1单克隆抗体抑制作用更显著。
2.2.2单克隆抗体抑制酶活性情况：将纯化的弓形虫RH株速殖子与单克隆抗体孵育后接种于细胞培养板中,以DTT刺激10分钟后加入Triton X-100,对于速殖子裂解液上清进行酶活性检测,观察单抗对酶活性的抑制作用。由于NTPase可水解ATP至ADP,同时释放出无机磷(Pi)。通过检测Pi浓度发现,与对照组相比,MNT1单克隆抗体处理组(P=0.002)与MNT2单克隆抗体处理组Pi浓度均显著降低(P=0.035),表明NTPase酶活性可被2株单克隆抗体显著抑制。
2.2.3单克隆抗体被动免疫保护性实验：将30只BALB/c小鼠随机分为6组,分别标记为MNT1-1、MNT1-2、MNT2-1、MNT2-2、IgG2a-1、IgG2a-2。将MNT1-1与MNT1-2两组BALB/c小鼠均以腹腔接种0.2ml(500μg/ml)单克隆抗体MNT1,MNT1-1组于腹腔接种单克隆抗体当天用200个RH株速殖子进入攻击感染,而MNT1-2组于接种48h后以同样数量速殖子攻击感染。MNT2组与IgG2a组根据组名接种不同单抗,其余操作步骤同上。密切观察、记录各组小鼠的死亡时间,评价单克隆抗体的被动免疫保护作用。结果表明,当天攻击感染的不同处理组间,其生存时间存在显著性差异(χ2=14.708,P=0.001),与对照组相比,MNT1单克隆抗体处理组(P=0.004)和MNT2单克隆抗体处理组(P=0.018)小鼠的生存时间均显著延长,而且,MNT1单克隆抗体处理组与MNT2单克隆抗体处理组中小鼠生存时间亦具有显著性差异(P=0.004);同样,48h后攻击感染的不同处理组间的生存时间亦存在显著性差异(χ2=6.531,P=0.038)。然而,尽管MNT1单克隆抗体处理组在48h后攻击感染其生存时间比对照组显著延长(P=0.030),MNT2单克隆抗体处理组与对照组之间无显著性差异(P=0.189),表明应用MNT1被动免疫后产生的免疫保护效果更好,可显著延长小鼠生存时间。本实验为进一步研发新弓形虫疫苗提供了实验依据。
3. rTgNTPase-Ⅱ对弓形虫感染的免疫保护及作用机制
通过观察rTgNTPase-Ⅱ免疫后,BALB/c小鼠体内所产生的免疫反应类型及免疫保护作用,为寻找新的疫苗靶点提供实验依据。将纯化的rTgNTPase-Ⅱ蛋白进行去内毒素处理,并通过凝胶法检测蛋白样品中内毒素含量,待样品中内毒素含量低于试剂盒最低检测量0.03EU/ml时对SPF级BALB/c小鼠进行免疫。小鼠分为3组：联合免疫组、rTgNTPase-Ⅱ单独免疫组以及对照组,每组各20只。其中联合免疫组以10μg的rTgNTPase-Ⅱ吸附0.5mg氢氧化铝佐剂进行联合免疫,对照组为PBS混合氢氧化铝佐剂进行免疫。初次免疫后每隔2周进行一次加强免疫,共加强免疫3次。待加强免疫结束后2周,对各免疫组小鼠进行特异性IgG抗体实验、细胞免疫反应实验及免疫保护作用研究。
3.1.体液免疫反应评价：
以5μg/ml的rTgNTPase-Ⅱ为包被抗原建立间接ELISA法,检测各免疫组小鼠血清中特异性总IgG抗体与IgG1、IgG2a两个抗体亚类。结果显示,联合免疫组中抗rTgNTPase-Ⅱ总IgG抗体比rTgNTPase-Ⅱ单独免疫组(P=0.000)和对照组(P=0.000)均显著升高,而rTgNTPase-Ⅱ单独免疫组与对照组相比亦显著升高(P=0.000)。此外,与对照组相比,联合免疫组小鼠血清中IgGl抗体亚类与IgG2a抗体亚类水平比rTgNTPase-Ⅱ单独免疫组(P=0.005,P=0.005)和对照组(P=0.000,P=0.000)均显著升高,而rTgNTPase-Ⅱ单独免疫组与对照组相比亦显著升高(P=0.000,P=0.000),提示rTgNTPase-Ⅱ所诱导产生的免疫反应可能为Th1/Th2混合型免疫反应。然而,通过计算各rTgNTPase-Ⅱ免疫组中IgG1与IgG2a比值,我们可以发现,rTgNTPase-Ⅱ单独免疫组中所诱导产生的为Thl型细胞免疫应答(IgG2a/IgG1>1),而联合免疫组中所诱导产生的为Th2型细胞免疫应答(IgG2a/IgG1<1),表明佐剂具有增强体液免疫反应的作用。
3.2.细胞免疫反应评价
3.2.1淋巴细胞增殖实验：最后一次加强免疫后2周,每组各取5只小鼠脾淋巴细胞,悬浮后按3×105/孔加入96孔细胞培养板,随后以rTgNTPase-Ⅱ(10μg/ml)刺激体外培养的免疫小鼠脾淋巴细胞,待孵育72小时后以CCK-8法检测各孔吸光度,按以下公式计算刺激指数：刺激指数(SI)=刺激孔均值/对照组均值。以ConA作为阳性对照,完全培养液作为阴性对照。与对照组相比,联合免疫组(P=0.000)与rTgNTPase-Ⅱ单独免疫组(P=0.002)中,小鼠脾淋巴细胞均对rTgNTPase-Ⅱ刺激出现了显著的淋巴细胞增殖反应,但两个免疫组之间差异并无显著性(P=0.295)。
3.2.2细胞因子检测：按上述方法得到小鼠脾淋巴细胞后,以5×106/孔铺入24孔细胞培养板,待孵育24小时后检测培养上清中IL2、IL-4浓度,72小时后检测IL-10浓度,96小时后检测IFN-γ浓度。与对照组相比,联合免疫组(P=0.000)与rTgNTPase-Ⅱ单独免疫组(P=0.000)中小鼠脾淋巴细胞均对rTgNTPase-Ⅱ刺激产生了IFN-γ。同样,联合免疫组(P=0.002)与rTgNTPase-Ⅱ单独免疫组(P=0.013)中小鼠脾淋巴细胞均对rTgNTPase-Ⅱ刺激产生了IL-2。然而却只有联合免疫组的细胞培养上清中IL-10含量与对照组(P=0.021)和rTgNTPase-Ⅱ单独免疫组(P=0.031)相比均出现显著升高,而所有免疫组中均未检测出IL-4。
3.2.3 T细胞亚群分析：按之前操作步骤分别获取各免疫组5只小鼠脾脏,制备脾细胞悬液,调整细胞浓度约106/50μ1/管,每管加入3株荧光标记的单克隆抗体：CD4(RM4-5)-FITC Rat Anti-Mouse、CD8a(53-6.7)-PE Rat Anti-Mouse和CD3e-PE-CYTM5 Hamster Anti-Mouse 20μl。暗室孵育后于流式细胞仪上计数10000个细胞,记录CD3+CD4+CD8"和CD3+CD4-CD8+细胞百分数。结果发现,联合免疫组(P=0.000)与rTgNTPase-Ⅱ单独免疫组(P=0.000)中CD3+CD4+双阳性T细胞/CD3+CD8+双阳性T细胞的比值均显著低于对照组。
3.3免疫保护作用评价
3.3.1急性攻击感染：为观察免疫小鼠对弓形虫急性感染的免疫保护作用,在免疫结束后2周用1000个弓形虫强毒株RH株速殖子于腹腔攻击感染各免疫组小鼠,每组各感染5只,记录各组小鼠生存时间。与对照组相比,联合免疫组(P=0.021)与rTgNTPase-Ⅱ单独免疫组(P=0.020)小鼠生存时间均显著延长。
3.3.2慢性攻击感染：为观察各免疫组小鼠对弓形虫慢性感染所产生的免疫保护作用,将各组最后剩下的10只小鼠以弓形虫弱毒株PRU株包囊经口喂食攻击感染,每只小鼠感染20个包囊。攻击感染5周后,计数各免疫组小鼠脑组织中包囊数。结果显示联合免疫组(P=0.008)与rTgNTPase-Ⅱ单独免疫组(P=0.014)脑组织包囊数均显著减少,下降率分别为62.9%和57.6%。从而证实rTgNTPase-Ⅱ可诱导BALB/c小鼠产生特异性免疫反应,该免疫反应以Thl型细胞应答为主。由rTgNTPase-Ⅱ诱导产生的免疫反应可为BALB/c小鼠提供部分的免疫保护作用。
Toxoplasma gondii is an obligate intracellular parasite and a significant pathogen of immunocompromised patients, notably persons with acquired immune deficiency syndromes (AIDS). Parasite replication and the strong inflammatory response result in massive tissue destruction and severe clinical manifestations. Although acute toxoplasmosis can be effectively treated with a variety of antibiotics, the drugs commonly cause side effects and treatment usually does not eradicate the infection. Therefore, new drugs, especially those against the proliferative stage of the parasite and with none or less side effect, are highly desirable for the treatment of toxoplasmosis.
T. gondii is surrounded with parasitophorous vacuole membrane after invasion of host cells. The parasite could modify the vacuolar environment by the secretion of a large number of proteins; one of them with dithiol-activated enzymatic activity is the nucleoside triphosphate hydrolase (NTPase). The NTPase is released from dense granules and accumulates as a soluble protein in the vacuolar space. The enzyme functions as an apyrase and is capable of degrading ATP to ADP and ADP to AMP. In host cells, T. gondii lacks the enzymes necessary for de novo synthesis of purines and therefore must salvage purines from the host for survival and replication, and NTPase has been proved to function in the purine salvage pathway by participating in the generation of the preferred purine salvage substrate, adenosine. So that it is supposed to take a significant role in parasite survival and replication.
There are two isoforms, NTPase-Ⅰand NTPase-Ⅱ, expressed in T. gondii. The genomic construction of the NTP genes has no intron and includes three tandemly repeated NTP genes:NTP1, NTP2 and NTP3, in which NTP1 encodes NTPase-Ⅱisoform, NTP3 encodes NTPase-Ⅰisoform and NTP2 does not encode protein. The gene encoding NTPase-Ⅱis found in all strains of T. gondii, while the gene encoding NTPase-Ⅰis confined only to virulent strains. As a result, the application of NTPase-Ⅱisoform may be more preferable as a potential vaccine candidate than NTPase-Ⅰisoform.
In this study, the monoclonal antibodies against recombinant T. gondii NTPase-Ⅱ(rTgNTPase-Ⅱ) were developed firstly, and then we undertook a series of experiments to test whether these McAbs inhibited the enzyme activity and the growth of T. gondii, and finally illustrate the effect of NTPase-Ⅱon growth of Toxoplasma gondii tachyzoite. Finally, in order to find novel T. gondii recombinant antigens with protective values, we describe here the ability of a recombinant form of TgNTPase-II co-administered with alum, a safe adjuvant that can be used in humans, to induce protection against toxoplasmosis in a murine model.
1. Clone and Expression of NTPase-Ⅱgene of Toxoplasma gondii
To construct a pBAD-HisB-NTPase recombinant plasmid and express the His-tag fusion protein NTPase, and identify the purified protein.
The NTPase gene was amplified by polymerase chain reaction (PCR) from RH strain of T.gondii and cloned into pGEM-T Easy vector. Positive clones were screened and identified by BglⅡ、HindⅢdigestion and sequenced. The homology of DNA sequence was 100% to that in the Genbank. The target gene was then subcloned into the prokaryotic expression vector pBAD-HisB and transformed into E.coli BL21 (DE3). So prokaryotic recombinant plasmids pBAD-HisB-NTPase were successfully constructed, SDS-PAGE showed that the recombinant NTPase proteins were over-expressed as inclusion body with molecular weight of 70 kDa.
The expressed recombinant proteins were purified with Ni-NTA agarose and further analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. Western blotting and ELISA analysis testified that the purified recombinant protein could be specifically recognized by mouse serum immunized with T. gondii and mouse anti-recombinant protein sera, and indicated the protein had highly antigenicity and immunogenicity.
2. Development and analysis of the monoclonal antibody against recombinant T. gondii NTPase-Ⅱ(rTgNTPase-II):
To develop the monoclonal antibodies against recombinant T. gondii NTPase-II (rTgNTPase-Ⅱ), undertake a series of experiments to test whether these McAbs inhibited the enzyme activity and the growth of T. gondii, and finally illustrate the effect of NTPase-Ⅱon growth of Toxoplasma gondii tachyzoite.
Syngeneic 6- to 8-week-old female BALB/c mice were injected subcutaneously with purified rTgNTPase-Ⅱantigen (50μg/100μl) mixed with an equal volume of Freund's complete adjuvant, and boosted 3 times subcutaneously with rTgNTPase-Ⅱ(50μg/100μl) mixed with Freund's incomplete adjuvant at 2 week intervals. Spleen cells obtained from immunized mice three days after the last immunization were fused with Sp2/0 myeloma cells by using polyethylene glycol. Supernatants from 534 hybridomas were screened respectively. Selection of positive hybridomas was made by ELISA using purified rTgNTPase-II as the antigen. Then, a positive hybridoma obtained was subcloned 3 times by limiting dilution. Finally, two McAb-producing hybridoma cell lines with the specific immunoactivity were identified and named as MNT1 and MNT2 respectively. Then, two McAb-producing hybridoma cell lines were transplanted intraperitoneally into BALB/c mice previously treated with pristane to collect ascites.
2.1. Characterization of McAbs:
At first, These McAbs isotypes were determined by Sigma kit and isotypes of both McAbs were IgG2a class. And titers of the culture supernatant and ascetic fluids of hybridoma cells were detected by indirect ELISA after serial dilutions. Results showed that the titers of MNT1 and MNT2 were 1:12800 and 1:6400 in culture supernatants; and 1:51200 and 1:12800 in ascitic fluids, respectively. Antibodies of cells were still positive after passage for three months and storage in liquid N2 for one month.
Then, these McAbs were purified on a protein A-agarose column and identified using SDS-PAGE and Western blotting. The BL21 (DE3) cell lysate containing pBAD-HisB was used as the negative control and whole tachyzoite lysate was used as positive controls. MNT1 and MNT2 can bind with the protein sized around 70 kDa and the protein sized around 63 kDa, respectively, but not with BL21 (DE3) cell lysate containing pBAD-HisB. The result displayed that the developed McAbs, MNTl and MNT2, can recognize the recombined and native NTPase-Ⅱspecifically.
Finally, in order to determine whether the two McAbs could react with NTPase-Ⅱprotein in tachyzoites, confocal laser microscopy was used for indirect fluorescence antibody test (IFAT) after immunostaining was carried out by treating tachyzoites with McAb-containing supernatants, both supernatants from Sp2/0 cultures and an isotype-matched mouse myeloma IgG2a as the negative control. Images showed that a specific reaction was observed on tachyzoites when T. gondii tachyzoites were analyzed with MNTl or MNT2 in IFAT.
2.2. Examination of the protective effects of McAbs:
COS-7 cells seeded in multiwell cell culture dishes were infected with tachyzoites (a parasite:cell ratio of 2:1). Prior to infection, tachyzoites were pre-incubated in 50μg/ml two McAbs for 1 h at 37℃, isotype-matched mouse myeloma IgG2a and medium as negative control. One hour after incubation, the monolayer was washed to remove extracellular parasites and re-incubated with medium for 2 h,8 h and 14 h, respectively. Cells on the coverslips were stained with Giemsa solution and examined by light microscopy. The number of infected cells per 100 cells and the number of tachyzoites per 100 infected cells were examined at 3 h after incubation for evaluation of the effects of McAbs on inhibition, and the number of tachyzoites per 100 infected cells was compared for evaluation of the effects of McAbs on replication at 3 h,9 h and 15 h after infection, respectively. The infective rates of MNT1, MNT2, IgG2a and medium treated tachyzoites were 12%,10%,12% and 13%, respectively and not significantly different among four groups(χ2= 0.458, P=0.928). However, there were significant differences in the number of intracellular parasites per infected cell between MNT1 or MNT2 treated group and IgG2a or medium treated group at 9h and 15h after infection. Pretreatment of tachyzoites with MNT1 or MNT2 significantly inhibited parasite replication in COS-7 compared to the IgG2a or medium control group at 9h (P=0.014, MNT1 versus medium; P=0.017, MNT1 versus IgG2a; P=0.037, MNT2 versus medium; P=0.040, MNT1 versus IgG2a) and 15h (P=0.000, MNT1 versus medium; P= 0.000, MNT1 versus IgG2a; P= 0.015, MNT2 versus medium; P=0.002, MNT1 versus IgG2a) after infection. Furthermore, the inhibition in tachyzoites pretreated with MNT1 McAb was significantly higher than those pretreated with MNT2 McAb (P= 0.003) at 15h after infection.
In order to examine the inhibition of NTPase activity by two McAbs in vitro, tachyzoites were pre-incubated with MNT1 or MNT2, respectively. After incubation, 1mM DTT was added and incubated for 10 min. Then Triton X-100 was added to a final concentration of 1%, and tachyzoites were incubated for 5 min at room temperature. The detergent lysates of tachyzoites were assayed for enzymatic activity. An isotype-matched mouse myeloma IgG2a was used as a negative control. The concentration of Pi reached 23.0±5.4μM in control group (P= 0.002, MNT1 versus IgG2a; P=0.035 MNT2 versus IgG2a), but it was 6.8±2.6μM in MNT1 group and 14.9±1.9μM in MNT2 group (P=0.036, MNT1 group versus MNT2 group), indicating that both MNT1 and MNT2 can inhibit the ATP diphosphohydrolase activity of NTPase in vitro, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the McAbs.
To evaluate the protection of passive immunization, two groups of BALB/c mice, MNT1-1 and MNT1-2, were immunized intraperitoneally with 0.2 ml (500μg/ml) of purified MNT1 McAb per mouse. Group MNT1-1 was challenged with two hundred RH strain tachyzoites on the same day of MNT1 injection. Group MNT1-2 was challenged with the same number of tachyzoites 48 h later. Mice in group MNT2-1 and MNT2-2 were injected with MNT2. The negative control groups A1 and A2 were injected with mouse myeloma IgG2a. The survival periods were recorded daily until all mice were dead. The mice, received MNT1 or MNT2 McAbs and challenged on the same day, displayed significantly longer survival times compared with the control group (P=0.004, MNT1 versus control; P=0.018, MNT2 versus control). On the other hand, although the survival time of mice receiving MNT1 then challenged after 48 h was significantly prolonged compared to the control (P=0.030), there was not significantly different between MNT2 group and control group (P=0.189). These results indicated that mice passively immunized with MNT1 survived considerably longer than other groups after challenge infection.
Taken together, we concluded that the McAbs against NTPase-Ⅱcan reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.
3. Study of the immune protection and the mechanism of rTgNTPase-Ⅱon T. gondii infection
In an effort to find novel T. gondii recombinant antigens with protective values, we describe here the ability of a recombinant form of TgNTPase-Ⅱco-administered with alum, a safe adjuvant that can be used in humans, to induce protection against toxoplasmosis in a murine model.
The recombinant NTPase-Ⅱprotein (rTgNTPase-Ⅱ) was expressed and purified. Endotoxin was removed. After endotoxin removal, the lipopolysaccharide (LPS) content was measured. Less than 10pg/ml of LPS was detected in the final protein preparations. Before inoculation into mice or stimulation in vitro, rTgNTPase-II was dialyzed against PBS, filtered throughout a 0.2μm-pore membrane and stored at-70℃. The purified recombinant protein was quantified by the Bradford method.
Then, female BALB/c mice 6-8 weeks old were divided into three groups with 20 mice per group. Mice were immunized with 10μg of rTgNTPase-Ⅱalone or adsorbed to 0.5mg of aluminum hydroxide gel (alum) by bilateral intramuscular injection into the quadriceps and boosted with the same dose three times per two-week interval. Another 20 mice received injection with PBS plus alum and were used as non-immunized controls. Two weeks after the last immunization, humoral and cellular immune responses were evaluated.
3.1. Evaluation of humoral immune response:
To determine the specific antibody titers, blood samples were obtained after the immunization schedule was completed, and assayed by an ELIS A with rTgNTPase-Ⅱas the bound target. The IgG antibodies against rTgNTPase-Ⅱwere significantly greater in sera of mice immunized with rTgNTPase-Ⅱ+alum (1.038±0.479) than those immunized with rTgNTPase-Ⅱalone (0.434±0.204) (P=0.000). As expected, mice immunized with PBS+alum did not present significant specific antibody titers (0.032±0.024).
In order to characterize whether a Thl and/or Th2 response was elicited in immunized mice, the distribution of IgG subtypes against rTgNTPase-Ⅱwas analyzed. Both IgG1 and IgG2a were found in the sera of mice vaccinated with rTgNTPase-Ⅱalone or with rTgNTPase-Ⅱ+alum, which showed a mixed anti-rTgNTPase-ⅡIgG1/IgG2a profile. However, immunization by rTgNTPase-Ⅱalone gave a Th1 response (IgG2a/IgG1 ratio>1), by rTgNTPase-Ⅱwith alum adjuvant appeared predominantly Th2 response (IgG2a/IgG1 ratio<1). These results indicate that the presence of alum enhances the specific humoral response.
3.2. Evaluation of cellular immune response
In order to study the antigen-specific lymphocyte responses induced by the vaccination, an in vitro lymphocyte proliferation assay was performed. Spleens were removed from 5 mice per group two weeks after the last booster injection under aseptic conditions and single-cell preparations were obtained. The cells were then plated at a density of 3×105 cells and cultured in the presence of rTgNTPase-Ⅱ(10μg/ml) or Concanavalin A (ConA; 5μg/ml; positive control) or medium alone (negative control). The plates were incubated for 72h and pulsed with 10μl of CCK-8 reagent. The stimulation index (SI=the mean OD450 values from recombinant antigen-stimulated cultures/the mean OD450 values from non-stimulated cultures) of each group was calculated. Both splenocytes from the mice immunized with adjuvant rTgNTPase-Ⅱ(P=0.000) and rTgNTPase-Ⅱalone (P=0.002) elicited a significant lymphocyte proliferative response to the rTgNTPase-Ⅱantigen stimulation when compared to the control group. Control mice vaccinated with PBS-alum did not respond to rTgNTPase-II stimulation. However, splenocytes from all immunized and control groups proliferated to comparable levels in response to the mitogen ConA.
To further characterize the immuno-modulation properties of this antigen, the concentration of cytokines in supernatants of spleen cells stimulated with rTgNTPase-II was analyzed. Spleen cells were obtained as described above and cultured. Supernatants from cultured splenocytes (5×106) were collected after 24,72 or 96 h of stimulation with rTgNTPase-Ⅱ(10μg/ml) and were analyzed for interleukin-2 (IL-2) and interleukin-4 (IL-4) activity at 24 h, for interleukin-10 (IL-10) activity at 72 h, and for gamma-interferon (IFN-γ) activity at 96 h. Both mice immunized with rTgNTPase-Ⅱ+alum and rTgNTPase-Ⅱalone produced a significant increase in the amount of secreted IFN-γ(P=0.000, rTgNTPase-Ⅱ+alum or alone group versus control) and IL-2 (P=0.002, rTgNTPase-Ⅱ+alum versus control; P=0.013, alone group versus control) when compared to the PBS+alum groups. Furthermore, only splenocytes of mice immunized by rTgNTPase-Ⅱ+alum produced specific amounts of IL-10 (P=0.021, rTgNTPase-Ⅱ+alum versus control; P=0.031, alone group versus control). In contrast, IL-4 was not detected in any group following stimulation with rTgNTPase-Ⅱantigen. In the meanwhile, Con A induced the highest cytokine release levels for these cytokines assayed in all experimental groups. These results show that immunization with rTgNTPase-Ⅱ+ alum induces a mixed Th1/Th2 response.
For phenotypic analysis of splenocytes, cell surface staining was accomplished by using the following fluorochrome-conjugated McAbs:anti-mouse-CD4-FITC, anti-mouse-CD8a-PE, anti-mouse-CD3e-PE-cy5 and appropriate IgG isotype controls. A single cell suspension was prepared as described above, then cells were delivered to each well already containing of CD4-FITC, CD8a-PE or CD3e-PE-Cy5 or appropriate IgG isotype controls and incubated. Erythrocytes were then lysed once. After washing, incubated cells were fixed and immediately analyzed on flow cytometer collecting at least 10,000 cells per sample. Although the percentages of CD3+CD4+ T lymphocytes were slightly higher in rTgNTPase-Ⅱ+alum and rTgNTPase-Ⅱalone groups than those in control group, there were not statistically significant differences (P=0.597). However, the ratio of CD3+CD4+ T cells/ CD3+CD8+ T cells was significantly reduced in all groups except the control group because of the significant increase of percentages of CD3+CD8+ T lymphocytes, suggesting that rTgNTPase-Ⅱ-induced immunity is also CD8+ T cell mediated.
3.3. Evaluation of protection against acute and chronic infection
To evaluate whether rTgNTPase-Ⅱcould potentially provide protection against T. gondii acute infection, five mice of each group were intraperitoneally challenged with 103 tachyzoites of the virulent RH strain 2 weeks after last immunization. A significant increase in the survival rate was observed in the rTgNTPase-Ⅱ+alum (P =0.021) and rTgNTPase-Ⅱalone immunized group (P=0.020) compared to control group, indicating that rTgNTPase-Ⅱcould induce partial protection against challenge with the highly virulent RH strain of T. gondii.
In addition to determining the protection against acute infection, rTgNTPase-Ⅱ-dependent resistance to T. gondii chronic infection was also evaluated. Ten mice of each group were inoculated orally with 20 PRU tissue cysts at two weeks after the last immunization. Mice were observed daily for mortality. Five weeks after the challenge, surviving mice were sacrificed and their brains were removed. The mean number of cysts per brain was determined by counting. Compared to the mice in control group, the average parasite burden was reduced significantly by 62.9% and 57.6% for rTgNTPase-Ⅱ+alum (P=0.008) and rTgNTPase-Ⅱalone vaccinated mice (P=0.014), respectively. These results demonstrated the protective effect of rTgNTPase-Ⅱagainst T. gondii chronic infection.
弓形虫侵入宿主细胞后位于一个由纳虫泡膜包绕的纳虫泡内,位于纳虫泡内的虫体可通过分泌一些蛋白成分修饰纳虫泡内环境,从而维持自身生长发育需求。其中,核苷三磷酸脱氢酶(nucleoside triphosphate hydrolase, NTPase)是弓形虫适应宿主细胞内环境获得嘌呤的寄生机制而产生,对弓形虫在寄生、繁殖方面具有重要意义。由于弓形虫属于嘌呤营养缺陷型,无法通过从头途径合成嘌呤碱基,因此,为了生存和繁殖,弓形虫只能通过补救合成途径直接从宿主细胞获取嘌呤。弓形虫NTPase蛋白存在于速殖子体膜表面,但当速殖子侵入宿主细胞不久,NTPase即分泌入纳虫泡的网状管腔系统内,在二巯基化合物(如DTT)的激活作用下,能连续水解所有的三磷酸核苷和三磷酸脱氧核苷至单磷酸形式。感染宿主细胞的弓形虫速殖子即是利用NTPase分解宿主细胞来源的ATP,以合成维持自身生存所必需的嘌呤核苷酸。
弓形虫NTPase蛋白有NTPase-Ⅰ和NTPase-Ⅱ两种亚型。编码NTPase基因组DNA无内含子,由3个连续排列的开放读码框架(ORF)组成,分别为NTP1、NTP2和NTP3。其中NTP2不具备编码蛋白的能力,而NTPase-Ⅰ、NTPase-Ⅱ是分别与编码基因NTP3和NTP1相对应的一组同工酶。其中NTPase-Ⅰ仅存在于有毒株,而NTPase-Ⅱ几乎存在于所有弓形虫虫株。显而易见,以NTPase-Ⅱ蛋白作为药物或疫苗靶点将比NTPase-Ⅰ蛋白具有更广泛地应用前景。
本研究将以原核表达的NTPase-Ⅱ蛋白免疫BALB/c小鼠,制备特异性抗弓形虫核苷三磷酸水解酶(NTPase)Ⅱ型重组蛋白(rTgNTPase-Ⅱ)单克隆抗体,观察所获单克隆抗体的保护作用,阐明弓形虫NTPase-Ⅱ在RH株速殖子生长发育中的作用。同时观察rTgNTPase-Ⅱ免疫后,BALB/c小鼠体内所产生的免疫反应类型及免疫保护作用,为寻找新的疫苗靶点提供实验依据。
1.弓形虫NTPase-Ⅱ的基因克隆和蛋白表达
采用PCR方法扩增得到刚地弓形虫RH株NTPase基因,克隆入pGEM-T Easy载体,经酶切与测序鉴定后亚克隆至表达质粒pBAD-HisB,成功构建原核表达载体pBAD-HisB-NTPase,转入E.coli BL21(DE3)中获得高效表达,表达产物主要存在于包涵体中。融合蛋白的相对分子量(Mr)约70 kDa,与理论值相近。
使用Ni离子亲和层析柱纯化重组质粒pBAD-HisB-NTPase表达产生的含组氨酸的重组蛋白,SDS-PAGE鉴定纯度约为84%. Western blotting和ELISA分析结果显示,纯化的重组蛋白可被鼠抗重组蛋白血清特异性识别,具有良好的免疫反应性和免疫原性。
2.抗弓形虫rNTPase-Ⅱ单克隆抗体的制备及其功能分析
通过制备特异性抗弓形虫核苷三磷酸水解酶(NTPase)Ⅱ型重组蛋白(rTgNTPase-Ⅱ)单克隆抗体,观察所获单克隆抗体的保护作用,以此阐明弓形虫NTPase-Ⅱ在RH株速殖子生长发育中的作用。
将rTgNTPase-Ⅱ抗原(50μg/100μl)以福氏完全佐剂等体积混合后皮下多点注射免疫BALB/c小鼠,并以同样剂量的rTgNTPase-Ⅱ抗原等体积混合福氏不完全佐剂后每隔2周进行1次加强免疫,一共进行3次加强免疫。最后一次加强免疫后3天取免疫小鼠脾细胞与小鼠骨髓瘤Sp2/0细胞进行细胞融合,细胞融合率为92.71%(534/576)。利用纯化的rTgNTPase-Ⅱ作为包括抗原,以间接ELISA法进行筛选。对于筛选到的阳性克隆株以有限稀释法进行3次亚克隆,最终得到2株高亲和力杂交瘤细胞系,分别命名为MNT1与MNT2。将得到的单克隆抗体注射经降植烷处理的BALB/c小鼠腹腔,收集腹水以亲和层析法进行单抗纯化。
2.1单克隆抗体的鉴定：
采用Sigma抗体亚型检测试剂盒测定单克隆抗体的免疫球蛋白类别和亚类,证实2株单克隆抗体均为IgG2a亚类。间接ELISA法测定杂交瘤细胞株培养上清与小鼠腹水效价,检测结果表明,MNT1与MNT2细胞培养上清效价分别为1：12 800 and 1：6 400,而小鼠腹水效价分别为1：51200与1：12 800。同时将杂交瘤细胞株连续传代三个月及液氮冻存一个月后复苏,不同时间取其培养液上清,用间接ELISA法检测其抗体效价,测定抗体分泌的稳定性。结果发现,细胞连续传代三个月,分泌抗体的能力稳定。液氮冻存一个月后复苏,对细胞上清进行检测,抗体仍为阳性。以Western Blot法鉴定单克隆抗体的特异性,结果表明,两株单克隆抗体与rTgNTPase-Ⅱ蛋白在约70kDa处出现反应条带,与弓形虫RH株速殖子全虫抗原在约63kDa处出现反应条带,均与预期结果相符。而与阴性对照(含pBAD-HisB载体的DE3细菌裂解液)之间未出现反应条带。最后,将纯化的弓形虫RH株速殖子分别与单克隆抗体或阴性对照反应后,加入FITC标记的羊抗鼠IgG,利用间接荧光抗体实验(IFAT)于激光共聚焦显微镜下观察NTPase-Ⅱ在速殖子中的定位,发现2株单克隆抗体均与弓形虫RH株速殖子出现荧光反应。
2.2单克隆抗体保护性实验
2.2.1单克隆抗体抑制虫体入侵与胞内生长发育情况：将纯化的弓形虫RH株速殖子与单克隆抗体孵育后按照虫株数：细胞数为2：1的比例,对COS-7宿主细胞进行体外细胞培养。并于感染后3小时、9小时与15小时分别取出不同处理组于油镜下进行计数观察。其中与单抗孵育3h的细胞培养孔中观察100个宿主细胞中被速殖子感染的细胞数量(感染率),评价单抗对弓形虫RH株速殖子入侵宿主细胞的抑制作用。同时分别计数与单抗孵育3h、9h、15h的细胞培养孔中100个感染速殖子的阳性细胞中弓形虫虫体数量(感染度),评价单克隆抗体对弓形虫RH株在宿主细胞内生长发育的抑制作用。感染3小时后的感染率检测结果表明：MNT1、MNT2、IgG2a与完全培养液处理后的感染率分别为12%,10%,12%与13%,各组之间结果无统计学差异(χ2=0.458,P=0.928),表明2株单克隆抗体无抑制弓形虫RH株速殖子入侵COS-7宿主细胞的能力。然而在速殖子感染9h(χ2=19.741,P=0.000)与15h(χ2=44.974,P=0.000)后,各处理组间感染度均存在显著性差异,其中MNT1单克隆抗体处理组抑制虫体在COS-7细胞中增殖的效果最好,MNT2单克隆抗体处理组次之。表明2株单克隆抗体均可显著抑制弓形虫RH株速殖子在宿主细胞内的生长发育。且MNT1单克隆抗体抑制作用更显著。
2.2.2单克隆抗体抑制酶活性情况：将纯化的弓形虫RH株速殖子与单克隆抗体孵育后接种于细胞培养板中,以DTT刺激10分钟后加入Triton X-100,对于速殖子裂解液上清进行酶活性检测,观察单抗对酶活性的抑制作用。由于NTPase可水解ATP至ADP,同时释放出无机磷(Pi)。通过检测Pi浓度发现,与对照组相比,MNT1单克隆抗体处理组(P=0.002)与MNT2单克隆抗体处理组Pi浓度均显著降低(P=0.035),表明NTPase酶活性可被2株单克隆抗体显著抑制。
2.2.3单克隆抗体被动免疫保护性实验：将30只BALB/c小鼠随机分为6组,分别标记为MNT1-1、MNT1-2、MNT2-1、MNT2-2、IgG2a-1、IgG2a-2。将MNT1-1与MNT1-2两组BALB/c小鼠均以腹腔接种0.2ml(500μg/ml)单克隆抗体MNT1,MNT1-1组于腹腔接种单克隆抗体当天用200个RH株速殖子进入攻击感染,而MNT1-2组于接种48h后以同样数量速殖子攻击感染。MNT2组与IgG2a组根据组名接种不同单抗,其余操作步骤同上。密切观察、记录各组小鼠的死亡时间,评价单克隆抗体的被动免疫保护作用。结果表明,当天攻击感染的不同处理组间,其生存时间存在显著性差异(χ2=14.708,P=0.001),与对照组相比,MNT1单克隆抗体处理组(P=0.004)和MNT2单克隆抗体处理组(P=0.018)小鼠的生存时间均显著延长,而且,MNT1单克隆抗体处理组与MNT2单克隆抗体处理组中小鼠生存时间亦具有显著性差异(P=0.004);同样,48h后攻击感染的不同处理组间的生存时间亦存在显著性差异(χ2=6.531,P=0.038)。然而,尽管MNT1单克隆抗体处理组在48h后攻击感染其生存时间比对照组显著延长(P=0.030),MNT2单克隆抗体处理组与对照组之间无显著性差异(P=0.189),表明应用MNT1被动免疫后产生的免疫保护效果更好,可显著延长小鼠生存时间。本实验为进一步研发新弓形虫疫苗提供了实验依据。
3. rTgNTPase-Ⅱ对弓形虫感染的免疫保护及作用机制
通过观察rTgNTPase-Ⅱ免疫后,BALB/c小鼠体内所产生的免疫反应类型及免疫保护作用,为寻找新的疫苗靶点提供实验依据。将纯化的rTgNTPase-Ⅱ蛋白进行去内毒素处理,并通过凝胶法检测蛋白样品中内毒素含量,待样品中内毒素含量低于试剂盒最低检测量0.03EU/ml时对SPF级BALB/c小鼠进行免疫。小鼠分为3组：联合免疫组、rTgNTPase-Ⅱ单独免疫组以及对照组,每组各20只。其中联合免疫组以10μg的rTgNTPase-Ⅱ吸附0.5mg氢氧化铝佐剂进行联合免疫,对照组为PBS混合氢氧化铝佐剂进行免疫。初次免疫后每隔2周进行一次加强免疫,共加强免疫3次。待加强免疫结束后2周,对各免疫组小鼠进行特异性IgG抗体实验、细胞免疫反应实验及免疫保护作用研究。
3.1.体液免疫反应评价：
以5μg/ml的rTgNTPase-Ⅱ为包被抗原建立间接ELISA法,检测各免疫组小鼠血清中特异性总IgG抗体与IgG1、IgG2a两个抗体亚类。结果显示,联合免疫组中抗rTgNTPase-Ⅱ总IgG抗体比rTgNTPase-Ⅱ单独免疫组(P=0.000)和对照组(P=0.000)均显著升高,而rTgNTPase-Ⅱ单独免疫组与对照组相比亦显著升高(P=0.000)。此外,与对照组相比,联合免疫组小鼠血清中IgGl抗体亚类与IgG2a抗体亚类水平比rTgNTPase-Ⅱ单独免疫组(P=0.005,P=0.005)和对照组(P=0.000,P=0.000)均显著升高,而rTgNTPase-Ⅱ单独免疫组与对照组相比亦显著升高(P=0.000,P=0.000),提示rTgNTPase-Ⅱ所诱导产生的免疫反应可能为Th1/Th2混合型免疫反应。然而,通过计算各rTgNTPase-Ⅱ免疫组中IgG1与IgG2a比值,我们可以发现,rTgNTPase-Ⅱ单独免疫组中所诱导产生的为Thl型细胞免疫应答(IgG2a/IgG1>1),而联合免疫组中所诱导产生的为Th2型细胞免疫应答(IgG2a/IgG1<1),表明佐剂具有增强体液免疫反应的作用。
3.2.细胞免疫反应评价
3.2.1淋巴细胞增殖实验：最后一次加强免疫后2周,每组各取5只小鼠脾淋巴细胞,悬浮后按3×105/孔加入96孔细胞培养板,随后以rTgNTPase-Ⅱ(10μg/ml)刺激体外培养的免疫小鼠脾淋巴细胞,待孵育72小时后以CCK-8法检测各孔吸光度,按以下公式计算刺激指数：刺激指数(SI)=刺激孔均值/对照组均值。以ConA作为阳性对照,完全培养液作为阴性对照。与对照组相比,联合免疫组(P=0.000)与rTgNTPase-Ⅱ单独免疫组(P=0.002)中,小鼠脾淋巴细胞均对rTgNTPase-Ⅱ刺激出现了显著的淋巴细胞增殖反应,但两个免疫组之间差异并无显著性(P=0.295)。
3.2.2细胞因子检测：按上述方法得到小鼠脾淋巴细胞后,以5×106/孔铺入24孔细胞培养板,待孵育24小时后检测培养上清中IL2、IL-4浓度,72小时后检测IL-10浓度,96小时后检测IFN-γ浓度。与对照组相比,联合免疫组(P=0.000)与rTgNTPase-Ⅱ单独免疫组(P=0.000)中小鼠脾淋巴细胞均对rTgNTPase-Ⅱ刺激产生了IFN-γ。同样,联合免疫组(P=0.002)与rTgNTPase-Ⅱ单独免疫组(P=0.013)中小鼠脾淋巴细胞均对rTgNTPase-Ⅱ刺激产生了IL-2。然而却只有联合免疫组的细胞培养上清中IL-10含量与对照组(P=0.021)和rTgNTPase-Ⅱ单独免疫组(P=0.031)相比均出现显著升高,而所有免疫组中均未检测出IL-4。
3.2.3 T细胞亚群分析：按之前操作步骤分别获取各免疫组5只小鼠脾脏,制备脾细胞悬液,调整细胞浓度约106/50μ1/管,每管加入3株荧光标记的单克隆抗体：CD4(RM4-5)-FITC Rat Anti-Mouse、CD8a(53-6.7)-PE Rat Anti-Mouse和CD3e-PE-CYTM5 Hamster Anti-Mouse 20μl。暗室孵育后于流式细胞仪上计数10000个细胞,记录CD3+CD4+CD8"和CD3+CD4-CD8+细胞百分数。结果发现,联合免疫组(P=0.000)与rTgNTPase-Ⅱ单独免疫组(P=0.000)中CD3+CD4+双阳性T细胞/CD3+CD8+双阳性T细胞的比值均显著低于对照组。
3.3免疫保护作用评价
3.3.1急性攻击感染：为观察免疫小鼠对弓形虫急性感染的免疫保护作用,在免疫结束后2周用1000个弓形虫强毒株RH株速殖子于腹腔攻击感染各免疫组小鼠,每组各感染5只,记录各组小鼠生存时间。与对照组相比,联合免疫组(P=0.021)与rTgNTPase-Ⅱ单独免疫组(P=0.020)小鼠生存时间均显著延长。
3.3.2慢性攻击感染：为观察各免疫组小鼠对弓形虫慢性感染所产生的免疫保护作用,将各组最后剩下的10只小鼠以弓形虫弱毒株PRU株包囊经口喂食攻击感染,每只小鼠感染20个包囊。攻击感染5周后,计数各免疫组小鼠脑组织中包囊数。结果显示联合免疫组(P=0.008)与rTgNTPase-Ⅱ单独免疫组(P=0.014)脑组织包囊数均显著减少,下降率分别为62.9%和57.6%。从而证实rTgNTPase-Ⅱ可诱导BALB/c小鼠产生特异性免疫反应,该免疫反应以Thl型细胞应答为主。由rTgNTPase-Ⅱ诱导产生的免疫反应可为BALB/c小鼠提供部分的免疫保护作用。
Toxoplasma gondii is an obligate intracellular parasite and a significant pathogen of immunocompromised patients, notably persons with acquired immune deficiency syndromes (AIDS). Parasite replication and the strong inflammatory response result in massive tissue destruction and severe clinical manifestations. Although acute toxoplasmosis can be effectively treated with a variety of antibiotics, the drugs commonly cause side effects and treatment usually does not eradicate the infection. Therefore, new drugs, especially those against the proliferative stage of the parasite and with none or less side effect, are highly desirable for the treatment of toxoplasmosis.
T. gondii is surrounded with parasitophorous vacuole membrane after invasion of host cells. The parasite could modify the vacuolar environment by the secretion of a large number of proteins; one of them with dithiol-activated enzymatic activity is the nucleoside triphosphate hydrolase (NTPase). The NTPase is released from dense granules and accumulates as a soluble protein in the vacuolar space. The enzyme functions as an apyrase and is capable of degrading ATP to ADP and ADP to AMP. In host cells, T. gondii lacks the enzymes necessary for de novo synthesis of purines and therefore must salvage purines from the host for survival and replication, and NTPase has been proved to function in the purine salvage pathway by participating in the generation of the preferred purine salvage substrate, adenosine. So that it is supposed to take a significant role in parasite survival and replication.
There are two isoforms, NTPase-Ⅰand NTPase-Ⅱ, expressed in T. gondii. The genomic construction of the NTP genes has no intron and includes three tandemly repeated NTP genes:NTP1, NTP2 and NTP3, in which NTP1 encodes NTPase-Ⅱisoform, NTP3 encodes NTPase-Ⅰisoform and NTP2 does not encode protein. The gene encoding NTPase-Ⅱis found in all strains of T. gondii, while the gene encoding NTPase-Ⅰis confined only to virulent strains. As a result, the application of NTPase-Ⅱisoform may be more preferable as a potential vaccine candidate than NTPase-Ⅰisoform.
In this study, the monoclonal antibodies against recombinant T. gondii NTPase-Ⅱ(rTgNTPase-Ⅱ) were developed firstly, and then we undertook a series of experiments to test whether these McAbs inhibited the enzyme activity and the growth of T. gondii, and finally illustrate the effect of NTPase-Ⅱon growth of Toxoplasma gondii tachyzoite. Finally, in order to find novel T. gondii recombinant antigens with protective values, we describe here the ability of a recombinant form of TgNTPase-II co-administered with alum, a safe adjuvant that can be used in humans, to induce protection against toxoplasmosis in a murine model.
1. Clone and Expression of NTPase-Ⅱgene of Toxoplasma gondii
To construct a pBAD-HisB-NTPase recombinant plasmid and express the His-tag fusion protein NTPase, and identify the purified protein.
The NTPase gene was amplified by polymerase chain reaction (PCR) from RH strain of T.gondii and cloned into pGEM-T Easy vector. Positive clones were screened and identified by BglⅡ、HindⅢdigestion and sequenced. The homology of DNA sequence was 100% to that in the Genbank. The target gene was then subcloned into the prokaryotic expression vector pBAD-HisB and transformed into E.coli BL21 (DE3). So prokaryotic recombinant plasmids pBAD-HisB-NTPase were successfully constructed, SDS-PAGE showed that the recombinant NTPase proteins were over-expressed as inclusion body with molecular weight of 70 kDa.
The expressed recombinant proteins were purified with Ni-NTA agarose and further analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. Western blotting and ELISA analysis testified that the purified recombinant protein could be specifically recognized by mouse serum immunized with T. gondii and mouse anti-recombinant protein sera, and indicated the protein had highly antigenicity and immunogenicity.
2. Development and analysis of the monoclonal antibody against recombinant T. gondii NTPase-Ⅱ(rTgNTPase-II):
To develop the monoclonal antibodies against recombinant T. gondii NTPase-II (rTgNTPase-Ⅱ), undertake a series of experiments to test whether these McAbs inhibited the enzyme activity and the growth of T. gondii, and finally illustrate the effect of NTPase-Ⅱon growth of Toxoplasma gondii tachyzoite.
Syngeneic 6- to 8-week-old female BALB/c mice were injected subcutaneously with purified rTgNTPase-Ⅱantigen (50μg/100μl) mixed with an equal volume of Freund's complete adjuvant, and boosted 3 times subcutaneously with rTgNTPase-Ⅱ(50μg/100μl) mixed with Freund's incomplete adjuvant at 2 week intervals. Spleen cells obtained from immunized mice three days after the last immunization were fused with Sp2/0 myeloma cells by using polyethylene glycol. Supernatants from 534 hybridomas were screened respectively. Selection of positive hybridomas was made by ELISA using purified rTgNTPase-II as the antigen. Then, a positive hybridoma obtained was subcloned 3 times by limiting dilution. Finally, two McAb-producing hybridoma cell lines with the specific immunoactivity were identified and named as MNT1 and MNT2 respectively. Then, two McAb-producing hybridoma cell lines were transplanted intraperitoneally into BALB/c mice previously treated with pristane to collect ascites.
2.1. Characterization of McAbs:
At first, These McAbs isotypes were determined by Sigma kit and isotypes of both McAbs were IgG2a class. And titers of the culture supernatant and ascetic fluids of hybridoma cells were detected by indirect ELISA after serial dilutions. Results showed that the titers of MNT1 and MNT2 were 1:12800 and 1:6400 in culture supernatants; and 1:51200 and 1:12800 in ascitic fluids, respectively. Antibodies of cells were still positive after passage for three months and storage in liquid N2 for one month.
Then, these McAbs were purified on a protein A-agarose column and identified using SDS-PAGE and Western blotting. The BL21 (DE3) cell lysate containing pBAD-HisB was used as the negative control and whole tachyzoite lysate was used as positive controls. MNT1 and MNT2 can bind with the protein sized around 70 kDa and the protein sized around 63 kDa, respectively, but not with BL21 (DE3) cell lysate containing pBAD-HisB. The result displayed that the developed McAbs, MNTl and MNT2, can recognize the recombined and native NTPase-Ⅱspecifically.
Finally, in order to determine whether the two McAbs could react with NTPase-Ⅱprotein in tachyzoites, confocal laser microscopy was used for indirect fluorescence antibody test (IFAT) after immunostaining was carried out by treating tachyzoites with McAb-containing supernatants, both supernatants from Sp2/0 cultures and an isotype-matched mouse myeloma IgG2a as the negative control. Images showed that a specific reaction was observed on tachyzoites when T. gondii tachyzoites were analyzed with MNTl or MNT2 in IFAT.
2.2. Examination of the protective effects of McAbs:
COS-7 cells seeded in multiwell cell culture dishes were infected with tachyzoites (a parasite:cell ratio of 2:1). Prior to infection, tachyzoites were pre-incubated in 50μg/ml two McAbs for 1 h at 37℃, isotype-matched mouse myeloma IgG2a and medium as negative control. One hour after incubation, the monolayer was washed to remove extracellular parasites and re-incubated with medium for 2 h,8 h and 14 h, respectively. Cells on the coverslips were stained with Giemsa solution and examined by light microscopy. The number of infected cells per 100 cells and the number of tachyzoites per 100 infected cells were examined at 3 h after incubation for evaluation of the effects of McAbs on inhibition, and the number of tachyzoites per 100 infected cells was compared for evaluation of the effects of McAbs on replication at 3 h,9 h and 15 h after infection, respectively. The infective rates of MNT1, MNT2, IgG2a and medium treated tachyzoites were 12%,10%,12% and 13%, respectively and not significantly different among four groups(χ2= 0.458, P=0.928). However, there were significant differences in the number of intracellular parasites per infected cell between MNT1 or MNT2 treated group and IgG2a or medium treated group at 9h and 15h after infection. Pretreatment of tachyzoites with MNT1 or MNT2 significantly inhibited parasite replication in COS-7 compared to the IgG2a or medium control group at 9h (P=0.014, MNT1 versus medium; P=0.017, MNT1 versus IgG2a; P=0.037, MNT2 versus medium; P=0.040, MNT1 versus IgG2a) and 15h (P=0.000, MNT1 versus medium; P= 0.000, MNT1 versus IgG2a; P= 0.015, MNT2 versus medium; P=0.002, MNT1 versus IgG2a) after infection. Furthermore, the inhibition in tachyzoites pretreated with MNT1 McAb was significantly higher than those pretreated with MNT2 McAb (P= 0.003) at 15h after infection.
In order to examine the inhibition of NTPase activity by two McAbs in vitro, tachyzoites were pre-incubated with MNT1 or MNT2, respectively. After incubation, 1mM DTT was added and incubated for 10 min. Then Triton X-100 was added to a final concentration of 1%, and tachyzoites were incubated for 5 min at room temperature. The detergent lysates of tachyzoites were assayed for enzymatic activity. An isotype-matched mouse myeloma IgG2a was used as a negative control. The concentration of Pi reached 23.0±5.4μM in control group (P= 0.002, MNT1 versus IgG2a; P=0.035 MNT2 versus IgG2a), but it was 6.8±2.6μM in MNT1 group and 14.9±1.9μM in MNT2 group (P=0.036, MNT1 group versus MNT2 group), indicating that both MNT1 and MNT2 can inhibit the ATP diphosphohydrolase activity of NTPase in vitro, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the McAbs.
To evaluate the protection of passive immunization, two groups of BALB/c mice, MNT1-1 and MNT1-2, were immunized intraperitoneally with 0.2 ml (500μg/ml) of purified MNT1 McAb per mouse. Group MNT1-1 was challenged with two hundred RH strain tachyzoites on the same day of MNT1 injection. Group MNT1-2 was challenged with the same number of tachyzoites 48 h later. Mice in group MNT2-1 and MNT2-2 were injected with MNT2. The negative control groups A1 and A2 were injected with mouse myeloma IgG2a. The survival periods were recorded daily until all mice were dead. The mice, received MNT1 or MNT2 McAbs and challenged on the same day, displayed significantly longer survival times compared with the control group (P=0.004, MNT1 versus control; P=0.018, MNT2 versus control). On the other hand, although the survival time of mice receiving MNT1 then challenged after 48 h was significantly prolonged compared to the control (P=0.030), there was not significantly different between MNT2 group and control group (P=0.189). These results indicated that mice passively immunized with MNT1 survived considerably longer than other groups after challenge infection.
Taken together, we concluded that the McAbs against NTPase-Ⅱcan reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.
3. Study of the immune protection and the mechanism of rTgNTPase-Ⅱon T. gondii infection
In an effort to find novel T. gondii recombinant antigens with protective values, we describe here the ability of a recombinant form of TgNTPase-Ⅱco-administered with alum, a safe adjuvant that can be used in humans, to induce protection against toxoplasmosis in a murine model.
The recombinant NTPase-Ⅱprotein (rTgNTPase-Ⅱ) was expressed and purified. Endotoxin was removed. After endotoxin removal, the lipopolysaccharide (LPS) content was measured. Less than 10pg/ml of LPS was detected in the final protein preparations. Before inoculation into mice or stimulation in vitro, rTgNTPase-II was dialyzed against PBS, filtered throughout a 0.2μm-pore membrane and stored at-70℃. The purified recombinant protein was quantified by the Bradford method.
Then, female BALB/c mice 6-8 weeks old were divided into three groups with 20 mice per group. Mice were immunized with 10μg of rTgNTPase-Ⅱalone or adsorbed to 0.5mg of aluminum hydroxide gel (alum) by bilateral intramuscular injection into the quadriceps and boosted with the same dose three times per two-week interval. Another 20 mice received injection with PBS plus alum and were used as non-immunized controls. Two weeks after the last immunization, humoral and cellular immune responses were evaluated.
3.1. Evaluation of humoral immune response:
To determine the specific antibody titers, blood samples were obtained after the immunization schedule was completed, and assayed by an ELIS A with rTgNTPase-Ⅱas the bound target. The IgG antibodies against rTgNTPase-Ⅱwere significantly greater in sera of mice immunized with rTgNTPase-Ⅱ+alum (1.038±0.479) than those immunized with rTgNTPase-Ⅱalone (0.434±0.204) (P=0.000). As expected, mice immunized with PBS+alum did not present significant specific antibody titers (0.032±0.024).
In order to characterize whether a Thl and/or Th2 response was elicited in immunized mice, the distribution of IgG subtypes against rTgNTPase-Ⅱwas analyzed. Both IgG1 and IgG2a were found in the sera of mice vaccinated with rTgNTPase-Ⅱalone or with rTgNTPase-Ⅱ+alum, which showed a mixed anti-rTgNTPase-ⅡIgG1/IgG2a profile. However, immunization by rTgNTPase-Ⅱalone gave a Th1 response (IgG2a/IgG1 ratio>1), by rTgNTPase-Ⅱwith alum adjuvant appeared predominantly Th2 response (IgG2a/IgG1 ratio<1). These results indicate that the presence of alum enhances the specific humoral response.
3.2. Evaluation of cellular immune response
In order to study the antigen-specific lymphocyte responses induced by the vaccination, an in vitro lymphocyte proliferation assay was performed. Spleens were removed from 5 mice per group two weeks after the last booster injection under aseptic conditions and single-cell preparations were obtained. The cells were then plated at a density of 3×105 cells and cultured in the presence of rTgNTPase-Ⅱ(10μg/ml) or Concanavalin A (ConA; 5μg/ml; positive control) or medium alone (negative control). The plates were incubated for 72h and pulsed with 10μl of CCK-8 reagent. The stimulation index (SI=the mean OD450 values from recombinant antigen-stimulated cultures/the mean OD450 values from non-stimulated cultures) of each group was calculated. Both splenocytes from the mice immunized with adjuvant rTgNTPase-Ⅱ(P=0.000) and rTgNTPase-Ⅱalone (P=0.002) elicited a significant lymphocyte proliferative response to the rTgNTPase-Ⅱantigen stimulation when compared to the control group. Control mice vaccinated with PBS-alum did not respond to rTgNTPase-II stimulation. However, splenocytes from all immunized and control groups proliferated to comparable levels in response to the mitogen ConA.
To further characterize the immuno-modulation properties of this antigen, the concentration of cytokines in supernatants of spleen cells stimulated with rTgNTPase-II was analyzed. Spleen cells were obtained as described above and cultured. Supernatants from cultured splenocytes (5×106) were collected after 24,72 or 96 h of stimulation with rTgNTPase-Ⅱ(10μg/ml) and were analyzed for interleukin-2 (IL-2) and interleukin-4 (IL-4) activity at 24 h, for interleukin-10 (IL-10) activity at 72 h, and for gamma-interferon (IFN-γ) activity at 96 h. Both mice immunized with rTgNTPase-Ⅱ+alum and rTgNTPase-Ⅱalone produced a significant increase in the amount of secreted IFN-γ(P=0.000, rTgNTPase-Ⅱ+alum or alone group versus control) and IL-2 (P=0.002, rTgNTPase-Ⅱ+alum versus control; P=0.013, alone group versus control) when compared to the PBS+alum groups. Furthermore, only splenocytes of mice immunized by rTgNTPase-Ⅱ+alum produced specific amounts of IL-10 (P=0.021, rTgNTPase-Ⅱ+alum versus control; P=0.031, alone group versus control). In contrast, IL-4 was not detected in any group following stimulation with rTgNTPase-Ⅱantigen. In the meanwhile, Con A induced the highest cytokine release levels for these cytokines assayed in all experimental groups. These results show that immunization with rTgNTPase-Ⅱ+ alum induces a mixed Th1/Th2 response.
For phenotypic analysis of splenocytes, cell surface staining was accomplished by using the following fluorochrome-conjugated McAbs:anti-mouse-CD4-FITC, anti-mouse-CD8a-PE, anti-mouse-CD3e-PE-cy5 and appropriate IgG isotype controls. A single cell suspension was prepared as described above, then cells were delivered to each well already containing of CD4-FITC, CD8a-PE or CD3e-PE-Cy5 or appropriate IgG isotype controls and incubated. Erythrocytes were then lysed once. After washing, incubated cells were fixed and immediately analyzed on flow cytometer collecting at least 10,000 cells per sample. Although the percentages of CD3+CD4+ T lymphocytes were slightly higher in rTgNTPase-Ⅱ+alum and rTgNTPase-Ⅱalone groups than those in control group, there were not statistically significant differences (P=0.597). However, the ratio of CD3+CD4+ T cells/ CD3+CD8+ T cells was significantly reduced in all groups except the control group because of the significant increase of percentages of CD3+CD8+ T lymphocytes, suggesting that rTgNTPase-Ⅱ-induced immunity is also CD8+ T cell mediated.
3.3. Evaluation of protection against acute and chronic infection
To evaluate whether rTgNTPase-Ⅱcould potentially provide protection against T. gondii acute infection, five mice of each group were intraperitoneally challenged with 103 tachyzoites of the virulent RH strain 2 weeks after last immunization. A significant increase in the survival rate was observed in the rTgNTPase-Ⅱ+alum (P =0.021) and rTgNTPase-Ⅱalone immunized group (P=0.020) compared to control group, indicating that rTgNTPase-Ⅱcould induce partial protection against challenge with the highly virulent RH strain of T. gondii.
In addition to determining the protection against acute infection, rTgNTPase-Ⅱ-dependent resistance to T. gondii chronic infection was also evaluated. Ten mice of each group were inoculated orally with 20 PRU tissue cysts at two weeks after the last immunization. Mice were observed daily for mortality. Five weeks after the challenge, surviving mice were sacrificed and their brains were removed. The mean number of cysts per brain was determined by counting. Compared to the mice in control group, the average parasite burden was reduced significantly by 62.9% and 57.6% for rTgNTPase-Ⅱ+alum (P=0.008) and rTgNTPase-Ⅱalone vaccinated mice (P=0.014), respectively. These results demonstrated the protective effect of rTgNTPase-Ⅱagainst T. gondii chronic infection.
引文
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