Construction and Evaluation of a Tuberculosis Multistage Vaccine Candidate Based on Recombinant Listeria ivanovii
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摘要:目的 构建以绵羊李斯特菌(Listerria ivanovii,LI)为载体的表达结核分枝杆菌(Mycobacterium tuberculosis,MTB)特征性抗原蛋白的疫苗候选菌株,并评价其生物安全性和免疫效果。方法 将MTB早期感染、潜伏感染和复发阶段的4种抗原基因的细胞表位串联形成融合抗原基因,即多阶段结核杆菌抗原基因,命名为msv。将msv插入含有LI同源序列的打靶质粒,利用同源重组技术构建基因组整合msv抗原基因的重组LI菌株。观察重组菌株的体外生长情况,通过Western blot验证靶抗原蛋白的表达情况,测定重组菌株对C57BL/6小鼠的半数致死量(50% lethal dose,LD50),以0.1×LD50为免疫剂量,通过尾静脉接种小鼠,通过接种前及接种后1、2、3、5、7、14 d的血清谷丙转氨酶(ALT)水平、脏器载菌量和脏器病理切片评价疫苗候选株的安全性。为测定疫苗候选株的免疫效果,另取3组小鼠分别尾静脉免疫LI-msv、LI、NS,免疫后第9天制备脾悬液,流式细胞术检测分泌干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、白细胞介素-2(IL-2)等细胞因子的CD4+ T细胞和CD8+ T细胞水平。结果 成功构建能表达多阶段结核杆菌抗原的疫苗候选株LI-msv。LI-msv 对C57BL/6小鼠的LD50为3.3×108 CFU/只。通过尾静脉接种小鼠后,LI-msv主要在小鼠肝脏和脾脏内短期繁殖,7 d后能被机体清除,对肝、脾的病理损伤时间短且可恢复;流式细胞术检测结果表明接种LI-msv的小鼠脾淋巴细胞分化出了特异的IFN-γ+ CD4+ T细胞和IFN-γ+ CD8+ T细胞,阳性细胞比率较相应载体对照组和生理盐水对照组高(P<0.005);特异性TNF-α+ CD4+ T细胞比率高于相应载体对照组(P<0.01)和生理盐水对照组(P<0.005),TNF-α+ CD8+ T细胞比率高于生理盐水对照组(P<0.005)。结论 成功构建了一株以LI为载体的表达结核杆菌多阶段抗原的疫苗候选株。该菌株具有生物安全性,且能诱导一定的特异性细胞免疫应答,有望作为结核疫苗候选株进行深入研究。Abstract:Objective To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model.Methods T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD50) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD50.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI-msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry.Results A recombinant strain named LI-msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD50 value of LI-msv for C57BL/6 mice (i.v.) was 3.3×108 CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ+ CD4+ and IFN-γ+ CD8+ T lymphocytes were successfully induced in LI-msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4+ and CD8+ T cells was significantly higher than those of vector control group and NS control group (P<0.005). Additionally, the frequency of specific TNF-α+ CD4+ T cell in LI-msv immunized group was significantly higher than that of vector control (P<0.01) and NS control group (P<0.005), and TNF-α+ CD8+ T cell frequency obviously increased than NS control group (P<0.005).Conclusions A novel multistage TB vaccine candidate expressing TB multistage antigen based on LI was successfully constructed. This vaccine candidate is safe and can induce specific cellular immune response to some extent. It is promising to be further studied as a candidate vaccine against tuberculosis.
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Keywords:
- Tuberculosis /
- Multistage tuberculosis vaccine /
- Listeria ivanovii /
- Safety /
- Immunogenicity
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由结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染引起的结核病(tuberculosis, TB)是严重威胁全球人类健康的重要公共卫生问题。MTB感染分为原发性感染、潜伏性感染(latent tuberculosis infection, LTBI)和原发后感染等状态,在不同感染状态,细菌的蛋白表达谱差异很大[1]。研究表明,选择MTB不同感染状态的特征性蛋白构建融合蛋白疫苗,可建立针对不同感染状态下MTB的特异性免疫应答,既能保护健康人群免受感染,又能有效控制清除LTBI人群和活动性TB患者体内的MTB[1]。
绵羊李斯特菌(Listerria ivanovii,LI)是典型的胞内寄生菌,对人不致病,具有低毒、安全的优点[2],已有研究者以LI为载体构建了携带MTB抗原的重组菌,免疫小鼠后,特异性细胞因子如干扰素-γ(IFN-γ)的分泌上调,初步验证了LI作为疫苗载体的可行性[3]。本研究选用MTB感染不同状态(早期感染、潜伏感染和复发阶段)的4种特征性抗原蛋白编码基因(Rv2660c、Rv2460、Rv3875和Rv3804c),筛选各抗原基因上T细胞表位,融合成多阶段MTB抗原基因,命名为msv,以LI作为疫苗活菌载体,将msv整合至其基因组特定位置,以此构建多阶段结核疫苗候选株,并评价该疫苗株对小鼠的安全性和免疫效果。
1. 材料与方法
1.1 材料
1.1.1 质粒、菌株、细胞、小鼠
质粒pUC57-msv携带融合抗原基因msv、酶切位点Hind Ⅲ和Xho Ⅰ,购自南京金斯瑞生物科技有限公司;中间质粒载体gst-402,含氨苄青霉素抗性基因(Ampr)、Hind Ⅲ、Xho Ⅰ和BamH Ⅰ酶切位点各1个;携带lacZ基因的重组绵羊李斯特菌 LI-lacZ,以上质粒和菌株由本实验室保存; E.coli DH5α感受态细胞,购自TaKaRa;C57BL/6小鼠,雌性,6~8周龄,清洁级别为SPF级,购自成都达硕生物科技公司。
1.1.2 PCR 引物
msv-f/r(f:5′-GTTCCAGATTATGCATCATTG-3′;r:5′-CCCAAACACGTGTATTATTCA-3′)用于筛选msv基因;Sosoo-f/r(f:5′-ATAATTTTGCTACTATGAAGGATCCATATCCATATGATGTTCCAG-3′;r:5′-TGGTCCATTTAATCCCTCGAGCCAACTATGTGTGCCACT-3′)用于扩增同源臂序列;红霉素(Ery)-f/r(f:5′-GTCGACGATTCACAAAAAATAGGC-3′;r:5′- ACTAGTCCCGGGGCGAATTG-3′)用于筛选Ery基因;LI-actA-f/r(f:5′-GAAGCTAAAAGTGCAAATGTCCC-3′;r:5′-ATTTCTTTAATACTGCGTTTGGGG-3′)用于筛选actA基因;LI-ldh- f/r(f:5′-GATTAAAGATTGGTGATTTAAAG-3′;r:5′- ACAAAAAACGGAAATCAGTTAG-3′)用于筛选LI菌株。以上引物由本实验室设计,并交由北京擎科新业生物技术有限公司合成。
1.1.3 主要仪器与试剂
质粒提取试剂盒 Plasmid Mini KitⅠ(200)、胶回收试剂盒E.Z.N.A.TM Gel Extraction Kit(200)购于OMEGA Bio-Tek;TreliefTM SoSoo Cloning Kit购于北京擎科新业生物技术有限公司;抗红细胞凝集表面抗原(HA)-小鼠IgG单克隆抗体、辣根过氧化物酶标记山羊抗小鼠IgG(H+L)购于碧云天生物技术有限公司;Bam HⅠ、Xho Ⅰ等限制性内切酶购于New England Biolabs;佛波脂(PMA)、离子霉素(Ionomycin)购于Sigma;Cytofix/Cytoperm试剂盒、PerCP 大鼠抗小鼠CD4、FITC 大鼠抗小鼠CD3、goligstop、perm wash(9×)购于BD PharMingenTM;PE 大鼠抗小鼠IFN-γ、PE-CyTM7 大鼠抗小鼠肿瘤坏死因子-α(TNF-α)、APC 大鼠抗小鼠白细胞介素-2(IL-2)购于Biolegend;PCR仪、ChemiDoc XRS凝胶成像系统购于BIO-RAD;流式细胞仪购于BD。
1.2 方法
1.2.1 MTB融合抗原基因msv的合成
从NCBI网站上获取Rv2460c、Rv2660c、Rv3804c和Rv3875的氨基酸序列,利用VaxiPred网上在线软件的4种计算方法,分别预测上述4个基因编码蛋白的辅助性T细胞(Th)抗原表位(http://www.imtech.res.in/raghava/propred)和细胞毒性T细胞(CTL)抗原表位(http://www.imtech. res.in/raghava/ctlpred)。将4个基因的T细胞表位通过柔性linker(GGACCAGGAGGC)连接,得到融合抗原基因msv,委托南京金斯瑞生物科技有限公司合成,提供克隆质粒pUC57-msv。
1.2.2 打靶质粒pCW612-msv的构建
将携带融合抗原基因的质粒pUC57-msv和中间质粒载体gst-402用Hind Ⅲ和XhoⅠ双酶切,通过连接转化构建中间质粒gst-msv。再利用BamH Ⅰ和XhoⅠ双酶切制备线性化克隆载体,同时通过PCR在插入片段两端分别引入与线性化载体两端一致的15~25 bp序列,利用同源重组的原理,将插入片段定向重组至载体中,构建打靶质粒pCW612-msv,PCR筛选阳性克隆菌并测序验证。
1.2.3 重组菌的构建与筛选
提取打靶质粒pCW612-msv,电转化LI-lacZ,将电转液涂布于含有Ery(3 μg/mL)、X-gal(8 mg/mL)和异丙基硫代半乳糖苷(IPTG,8 mg/mL)的脑心浸液琼脂平板(brain-heart infusion agar,BHI琼脂),30 ℃培养48 h,挑取蓝色菌落进行PCR验证和提取质粒验证。电转成功的菌落通过42 ℃和Ery抗性压力(3 μg/mL)实现第一次同源重组,30 ℃传代培养6代完成第二次同源重组获得重组菌,用相应引物对抗Ery基因、靶抗原盒、ldh、actA基因进行PCR验证和测序鉴定,将测序验证完全正确的阳性菌命名为LI-msv。
1.2.4 体外生长情况
将LI、LI-msv接种于适量BHI肉汤,37 ℃ 180 r/min摇床培养,以BHI肉汤为空白调零,每隔1 h测定一次菌液在600 nm处的吸光度值(A600)直至第9小时,得到重组菌和野生株的体外生长曲线。
1.2.5 Western blot法验证LI-msv靶抗原蛋白 msv
用BHI液体培养基培养LI-msv至对数生长期,收集培养上清和细菌沉淀,采用超声破壁、TCA-丙酮沉淀法提取蛋白。所有样品经SDS-PAGE电泳,以抗HA-小鼠IgG单克隆抗体为一抗,辣根过氧化物酶标记山羊抗小鼠IgG(H+L)为二抗,对转印且封闭后的PVDF膜进行孵育,孵育结束后,用HRP化学发光底物显色,分析。
1.2.6 半数致死剂量(LD50)的测定
C57BL/6小鼠随机分为4组,每组10只,尾静脉分别接种1.2×108、3.0×108、7.5×108和1.9×109 CFU/只的LI-msv,接种后连续10 d观察小鼠的生存情况,绘制生存曲线,根据改良寇式法计算LI-msv在尾静脉注射条件下对C57BL/6小鼠的LD50。
1.2.7 安全性评价
以0.1×LD50为免疫剂量,通过尾静脉接种小鼠(n=21),分别在小鼠接种前和接种后的1、2、3、5、7和14 d,随机选择3只小鼠眼眶取血、处死,无菌取出肝、脾,利用0.1% Triton X-100进行匀浆,匀浆后进行10倍系列稀释,取稀释液进行菌落计数,根据稀释度计算各脏器中的细菌载量(lgCFU/organ);同时测定血清中谷丙转氨酶(ALT)含量的变化并通过HE染色观察接种前及接种后1、3、14 d主要靶器官(肝、脾)的病理变化。
1.2.8 免疫效果评价
C57BL/6小鼠随机分为3组(每组10只,重复3次):阴性对照组(NS组);载体对照组(LI-lacZ组);实验组(LI-msv组)。以0.1×LD50为免疫剂量,通过尾静脉接种小鼠,接种后第9天处死,无菌取出脾脏,经研磨、裂红、计数处理后铺板,加入特异性肽段进行刺激并孵育,以含10%PBS的1640培养基作为未刺激的本底对照。用流式细胞术分析脾细胞中分泌IFN-γ、TNF-α、IL-2等细胞因子的CD4+ T细胞和CD8+ T细胞比率。
2. 结果
2.1 打靶质粒pCW612的验证
pCW612的PCR筛选和酶切验证结果如图1所示,以引物msv-f/r 进行PCR得到1 006 bp的条带;以引物LI-ldh-f/r 进行PCR得到1 012 bp的条带; BamH Ⅰ和XhoⅠ双酶切后分别得到8 946 bp和1 028 bp的目的条带。上述PCR及酶切结果均与预期结果相符,且测序验证该序列无误,提示打靶质粒pCW612构建成功。
图 1 打靶质粒pCW612的PCR筛选(A)和酶切验证(B)Figure 1. PCR screening (A) and restriction enzyme digestion (B) of targeting plasmid pCW612M: DL2000 DNA marker; 1: Amplification of msv from pCW612 with primers msv-f/r; 2: Amplification of LI-ldh from pCW612 with primers LI-ldh-f/r; 3: pCW612; 4: The product from pCW612 digested with Bam HⅠ and Xho Ⅰ2.2 重组菌筛选
同源重组后对重组菌进行PCR验证,结果见图2A:以引物Ery-f/r进行PCR筛选,在1 463 bp处无明显条带;以引物msv-f/r进行PCR筛选,在1 006 bp处有明显条带;以引物LI-actA-f/r进行PCR筛选,在635 bp处有明显条带。以上PCR结果与预期相符,测序结果也与预期相符。以上结果表明,同源重组后多阶段结核抗原基因已整合至LI基因组中,且打靶质粒已丢失,说明重组菌株构建成功。重组菌和野生菌的体外生长曲线(图2B)表明两者生长情况趋同;重组菌Western blot(图2C)可见体外培养至对数生长期后,重组菌能够在培养液上清和菌体裂解液中检测到相应抗原蛋白msv,表明重组菌能成功表达靶抗原。
图 2 重组菌PCR验证结果(A)、生长曲线(B)和对数生长期时靶蛋白的表达情况(C)Figure 2. Genome PCR (A), growth curve (B) and Western blot result (C) of recombinant strain LI-msvM: DL2000 DNA marker; 1: Amplification of Ery from LI-msv with primers Ery-f/r; 2: Amplification of msv from LI-msv with primers msv-f/r;3: Amplification of actA from LI-msv with primers LI-actA-f/r; NC: Negative control; PC: Positive control; Supernatant: Protein extracted from the culture supernatant; Cell lysate: Protein extracted from cell lysate2.3 外源基因的插入对重组菌株毒力的影响
观察接种了不同剂量的疫苗候选株的C57BL/6小鼠10 d的生存曲线(图3),根据改良寇氏法计算可知LI-msv对C57BL/6小鼠的LD50为3.3×108 CFU/只。
2.4 安全性评价
以0.1×LD50为免疫剂量通过尾静脉接种小鼠后,小鼠的肝脏(图4A)和脾脏(图4B)载菌量均在接种后第1天或第2天达到峰值,随后逐渐降低,第3天或第7天完全从脏器中清除,第14天没有复燃迹象。ALT含量(图4C)在第1天达到最高水平,随后迅速降至接种前水平。
图 4 C57BL/6小鼠尾静脉接种LI-msv后肝脏(A)、脾脏(B)载菌量,血清ALT水平(C)和脏器病理变化(D,HE ×200)Figure 4. CFU titers of recombinant strain in C57BL/6 mice liver (A) and spleen (B), serum ALT level (C) and pathological changes of organs (D,HE ×200) after i.v. inoculation*P<0.05, vs. 0 d; Dash lines in A and B: Minimum detection limit of counting method; Black arrows in D indicate the location of the lesion脏器病理变化情况如图4D所示(仅显示部位特点图片):第1天肝脏出现片状坏死;脾小结淋巴细胞大量减少,红髓中可见大量红细胞,可见少量多核巨细胞及中性粒细胞浸润。第3天肝损伤最为严重,除水肿外,汇管周围可见炎症细胞灶性浸润;脾脏红髓静脉中存在大量大块血栓,部分血管中可见散落的大量炎症细胞。第14天肝细胞除有少量炎症细胞浸润外基本恢复正常;脾脏组织恢复正常,红白髓界限分明。
2.5 免疫效果评价
见图5。接种疫苗候选株的小鼠与接种载体菌对照和NS对照的小鼠相比,其脾淋巴细胞中分泌IFN-γ的CD4+ T细胞、CD8+ T细胞的比率增加,差异有统计学意义(P<0.005);分泌TNF-α的CD4+ T细胞的比率高于载体菌对照组(P<0.01)和NS组(P<0.005),分泌TNF-α的CD8+ T细胞的比率仅与NS组相比差异统计学意义(P<0.01),与载体菌对照组相比有所上调,但差异无统计学意义(P>0.05);分泌IL-2的CD4+ T细胞、CD8+ T细胞的比率变化不明显,3组间差异无统计学意义(P>0.05)。
3. 讨论
TB是严重威胁全球人类健康的公共卫生难题之一,卡介苗作为全球范围内唯一有效并广泛使用的疫苗,对成人来说保护效果不理想。大量流行病学调查研究表明,卡介苗在不同地区不同人群中的保护效果在0~80%不等,且其保护效力只能持续10~15年[4]。此外,当宿主免疫力下降时,LTBI者体内处于休眠状态的MTB可能被激活,导致TB复发。因此研制新型结核疫苗变得尤为重要。
有研究者提出了兼具预防和治疗功效的多阶段疫苗的研发思路[1]。本研究选择MTB不同感染状态的特异性抗原蛋白编码基因连接融合作为多阶段MTB抗原基因。首先选择的是两种经典的指示活动性TB的抗原编码基因Rv3875和Rv3804c。研究表明,由Rv3875编码的早期分泌抗原靶6 kDa蛋白(early secretory antigenic target of 6 kDa, ESAT-6)能诱导强烈的T细胞免疫应答,且与Ag85B协同作用可以产生更好以及有效的免疫保护[5]。由Rv3804c编码的Ag85A的分泌率较高,可使细胞毒性T细胞的细胞毒活力升高,并刺激机体分泌IFN-γ、TNF-α和IL-2等细胞因子[6]。Rv2660c是MTB的一种压力响应基因,其编码的蛋白在MTB处于休眠状态时仍能持续表达。研究表明其能够诱导针对潜在MTB的免疫反应[7],提示Rv2660c基因可作为清除潜伏性感染阶段MTB的作用靶点,其引起的促炎反应可能是维持MTB潜伏期的重要途径。此外,有研究证实将Rv2660c基因与Ag85B-ESAT-6的结核重组蛋白疫苗H1融合,构建新型H56结核蛋白疫苗,可以提高H1免疫保护效果,还可以用于LTBI的控制,协助机体清除休眠状态的MTB[1]。除了以上3个基因,我们还选用了Rv2460c基因。Rv2460c基因参与LTBI阶段MTB代谢的调控,体外模拟LTBI的低氧低营养状态,证实其编码的clpP蛋白水解酶亚单位2(clpP2)在低氧状态下能持续性表达,而且,当MTB从低氧状态恢复到正常状态时,clpP2的表达量显著性提高,表明Rv2460c基因可用于提示TB复发[8-9]。
本研究选择低毒、安全的LI作为结核疫苗活菌载体,通过基因连接、质粒转化、同源重组等技术得到一株携带并表达多阶段MTB抗原的LI疫苗候选株,在C57BL/6小鼠模型中证实其具有生物安全性且能诱导一定的特异性免疫应答。李斯特菌感染机体后被吞噬细胞摄入,进入胞浆中增殖,再被邻近细胞摄入,此细胞内寄生和细胞间直接转染的特点,保证了李斯特菌能引起高效的特异性 T 细胞免疫应答。此外,MTB感染机体后,主要通过特异性细胞免疫应答将其清除,因此评价此多阶段结核疫苗候选株的免疫效果时仅从细胞免疫方面进行检测。疫苗候选株通过尾静脉免疫后,主要诱导分泌IFN-γ的T细胞,其诱导分泌特异性TNF-α和IL-2的T细胞的能力较弱。这可能与单次免疫效果有限有关,后续可采用初免-加强免疫策略。目前,在以李斯特菌为载体的疫苗研究中,均是采用单一的单增李斯特菌(Listerria monocytogenes,LM)或LI载体疫苗进行初次或加强免疫[10-13]。然而,使用单一疫苗初次和加强免疫,可能导致抗载体效应,削弱免疫保护效果。因此,在本研究构建的LI疫苗候选株基础上,可进一步开展初免-异种加强免疫策略,例如将本疫苗作为卡介苗的增强疫苗,后续可继续开展相关研究,拓宽以李斯特菌为载体的疫苗研究的发展。
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图 1 打靶质粒pCW612的PCR筛选(A)和酶切验证(B)
Figure 1. PCR screening (A) and restriction enzyme digestion (B) of targeting plasmid pCW612
M: DL2000 DNA marker; 1: Amplification of msv from pCW612 with primers msv-f/r; 2: Amplification of LI-ldh from pCW612 with primers LI-ldh-f/r; 3: pCW612; 4: The product from pCW612 digested with Bam HⅠ and Xho Ⅰ
图 2 重组菌PCR验证结果(A)、生长曲线(B)和对数生长期时靶蛋白的表达情况(C)
Figure 2. Genome PCR (A), growth curve (B) and Western blot result (C) of recombinant strain LI-msv
M: DL2000 DNA marker; 1: Amplification of Ery from LI-msv with primers Ery-f/r; 2: Amplification of msv from LI-msv with primers msv-f/r;3: Amplification of actA from LI-msv with primers LI-actA-f/r; NC: Negative control; PC: Positive control; Supernatant: Protein extracted from the culture supernatant; Cell lysate: Protein extracted from cell lysate
图 4 C57BL/6小鼠尾静脉接种LI-msv后肝脏(A)、脾脏(B)载菌量,血清ALT水平(C)和脏器病理变化(D,HE ×200)
Figure 4. CFU titers of recombinant strain in C57BL/6 mice liver (A) and spleen (B), serum ALT level (C) and pathological changes of organs (D,HE ×200) after i.v. inoculation
*P<0.05, vs. 0 d; Dash lines in A and B: Minimum detection limit of counting method; Black arrows in D indicate the location of the lesion
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