WT1抗原多表位基因疫苗设计及免疫原性研究

【作者】 田卫伟；

【导师】 乔振华；

【作者基本信息】 山西医科大学 ， 内科学， 2011， 博士

【摘要】 目的WT1（Wilms’tumor gene 1）是最早发现与Wilms’肿瘤发生发展有关的基因,在成人仅在少量的正常组织表达,但在各型白血病、骨髓异常增生综合征及多种实体瘤呈高表达,并与疾病的不良预后有相关性。反义寡核苷酸抑制WT1表达,致使白血病细胞及实体瘤细胞生长停止,以及WT1能诱导特异性的细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）反应均提示WT1可作为广谱性免疫治疗的理想靶原。迄今,基于WT1的表位肽疫苗已进入临床试验,并初见成效。然而目前研究者多采用单一CTL表位的疫苗,难免免疫原性差,且由于T细胞介导的免疫应答受主要组织相容性复合物（major histocompatibility complex,MHC）限制性,某一WT1的表位疫苗可能仅仅适用于少部分人类白细胞抗原（human leukocytes antigen,HLA）相合的患者,因此该疫苗的应用受到限制。构建多表位WT1的核酸疫苗可以弥补上述缺点,其优越性表现为:①内源性表位提呈可避免表位肽的降解,产生持久的细胞和体液免疫应答;②同时构建WT1的多个优势表位或受不同HLA限制性的表位,可有效应防止免疫逃逸和突破HLA限制;③同时构建WT1辅助性T细胞（helper T cell,Th）表位,其激发的CD4+T细胞,不仅可以更有效的激发和维持CTL免疫应答,而且可以产生特异性的抗肿瘤应答^[1] ;④易于设计和构建,且制备和贮存方便。此外,根据具体需要,可以将免疫调节因子构建于载体使其共表达,诱导产生最适免疫应答。因此,多表位基因疫苗在WT1抗原免疫治疗中具有广泛应用前景。基因佐剂是提高基因疫苗免疫效果的有效手段,研究表明,结核分枝杆菌热休克蛋白HSP70（mHSP70）是良好的免疫佐剂,其羧基端HSP70_359-610是其有效佐剂部位,可与CD40分子结合,通过p38信号通路诱导DC的成熟和极化Th1反应。而HSP70_407-426是位于mHSP70多肽结合区p359-610的一个刺激表位,选择该区域能比HSP70更显著的刺激DCs成熟,且这种作用不会被HSP70抑制表位p457-496所抑制^[2]。因此,mHSP70_407-426作为天然免疫佐剂在基因疫苗开发中具有潜在应用价值。经查阅文献,目前国内外尚未见有WT1多表位基因疫苗的报道,本文拟利用基因工程的方法将WT1多个CTL和Th细胞表位克隆至真核表达质粒pcDNA3.1中,构建WT1抗原多表位基因疫苗pcDNA-WT1。为获得更好的免疫效果,同时以mHSP70的刺激表位p407-426作为免疫佐剂,尝试构建WT1多表位与热休克蛋白70刺激表位融合基因疫苗pcDNA-WT1-HSP70_407-426,并探讨WT1抗原多表位基因疫苗及融合基因疫苗在小鼠诱导免疫应答的能力,研究其对荷瘤小鼠的抑瘤效应。此外,利用pcDNA-WT1-HSP70_407-426转染的HLA-A*0201⁺外周血单个核细胞（PBMC）诱导自身淋巴细胞产生CTL,通过乳酸脱氢酶释放法检测HLA-A*0201⁺限制性的CTL反应。方法1 WT1多表位盒的设计与合成本研究在收集已发表WT1表位的基础上,筛选具有良好免疫原性的WT1 CTL与Th表位进行表位盒组建,并采用理论结合软件模拟的方法,通过蛋白酶体切割软件（PAProC和NetChop）和DNA疫苗在线预测工具（DyNAVacS）优化表位盒构成后进行人工合成。2 WT1多表位基因疫苗的设计与构建2.1质粒构建将WT1多表位片段插入T载体构建质粒pUC57-WT1,经测序验证后,进行酶切获得WT1多表位片段,然后将该片段克隆至真核表达质粒pcDNA3.1中,构建重组质粒pcDNA-WT1; PCR扩增pGEM-mHSP70质粒HSP70中刺激表位片段p407-426,插入真核表达质粒pcDNA3.1,构建质粒pcDNA-HSP70_407-426;将上述获得的WT1多表位片段酶切插入质粒pcDNA-HSP70_407-426刺激表位的上游,构建质粒pcDNA-WT1-HSP70_407-426。上述质粒经酶切、PCR和测序鉴定。2.2转染293T和Hela细胞脂质体介导法瞬时转染293T细胞和Hela细胞,同时共转染pcDNA-EGFP荧光质粒,激光共聚焦显微镜下观察转染效果。2.3 RT-PCR和Westernblot验证转染293T和Hela细胞48小时后,提取293T细胞总RNA,RT-PCR检测多表位WT1和融合基因片段的表达;提取Hela细胞蛋白,Western blot法检测WT1相应功能蛋白的表达。3 WT1多表位基因疫苗的免疫原性研究3.1动物免疫C57BL/6雄性4-6周龄小鼠25只,随机分为5组,即PBS对照组、空质粒对照组、pcDNA- HSP70_407-426对照组、pcDNA-WT1实验组和pcDNA-WT1-HSP70_407-426实验组,每组5只。每只小鼠分别于0.25%盐酸布比卡因预处理双侧股四头肌48h后,注射质粒共50μg。每间隔2周注射一次,共免疫3次。最后一次免疫后10天,分离小鼠脾脏淋巴细胞。3.2免疫指标检测分离小鼠脾脏淋巴细胞后,通过MTT法检测小鼠T淋巴细胞增殖、流式细胞仪检测CD4+/CD8+T淋巴细胞亚群、ELISPOT法检测WT1特异性的IFN-γ分泌频数和LDH法检测杀伤性T淋巴细胞（CTL）功能。3.3疫苗对肿瘤的治疗作用4-6周龄C57BL/6小鼠25只,每组5只,分别在左前肢皮下接种高表达WT1的FBL3细胞（2x106/只）。7d后,小鼠肌肉接种DNA疫苗,注射质粒共50μg,对照组注射PBS、空质粒和pcDNA- HSP70_407-426。14d后加强免疫一次。待可触及肿瘤后,测量肿瘤体积,记录小鼠生存活动情况和存活率。3.4 HLA-A*0201限制性CTL反应分离健康HLA-A*0201患者外周血单个核细胞（PBMC）,将其中一部分转染pcDNA-WT1-HSP70_407-426后,与未转染部分做混合淋巴细胞培养诱导自身淋巴细胞产生CTL,通过通过乳酸脱氢酶释放法检测HLA-A*0201限制性的CTL反应。结果1 WT1多表位盒的设计与合成1.1表位肽的选取选择的WT1表位除能诱发良好的CTL或/和Th反应外,需尽量能与更多MHC分子结合。被选取的CTL表位有:WT1.37（VLDFAPPGA;HLA-A*0201）、WT1.187（SLGEQQYSV;HLA-A*0201/HLA-A*0206）和WT1.235（CMWNQMNL;HLA-A*0201/HLA-A*2402）,Th表位有:WT1.121A（SGQAYMFPNAPYLPSCLES;HLA-DRB1*0401）和WT1.332（KRYFKLSHLQMHSRKH;HLA-DRB1*0405/HLA-DRB1*1501/HLA-DRB1*1502/HLA-DPB1*0901）,其中WT1.121A中隐藏CTL表位WT1.126m（下划线,HLA-A*0201/H2Db）,此外,为了使构建的表位能更好的突破MHC限制性,同时构建了通用Th表位PADRE（KFVAAWTLKAAA）。1.2表位盒的优化与合成为了使构建的表位能够发挥各自独立的功能,不同的CTL表位之间加入蛋白酶体切割喜好位点如AAY、KK、KAA、KAAA、AAA和NAAA,并根据蛋白酶体切割预测软件PAProC和NetChop优化各表位间间隔序列。不同的Th表位采用柔性连接表位“GPGPG”进行连接。在此基础上加入内质网信号引导序列IgK以促进蛋白分泌,并加入包含ATG启动信号和KOZAK序列,以引导正确的翻译。通过在线预测工具DyNAVacS将得到的核苷酸序列按照真核偏爱密码子进行优化,之后人工合成,最终获得理论上具有良好功能的WT1表位盒。2 WT1多表位基因疫苗的设计与构建2.1质粒构建经酶切、PCR和测序鉴定,pcDNA-WT1、pcDNA-HSP70_407-426和pcDNA-WT1-HSP -70_407-426构建成功。2.2脂质体转染重组质粒转染细胞24小时后,观察绿色荧光蛋白表达,结果未转染的对照细胞无荧光出现,而转染重质粒的细胞有荧光出现,表明重组质粒能够在真核细胞中成功表达。2.3 RT-PCR和Westernblot验证提取转有质粒pcDNA-WT1、pcDNA-HSP70_407-426和pcDNA-WT1-HSP70_407-426的293T细胞总RNA,经RT-PCR扩增得到WT1和HSP70_407-426各基因片段,证实经质粒载体携带的基因及重组基因均已在真核生物中得到正确转录;提取转染有pcDNA-WT1-HSP70_407-426质粒的Hela细胞总蛋白,WT1作为一抗,Western blot检测结果表明,重组质粒在相对分子质量约29 000KD处有一特异性的蛋白反应带,与预期大小一致。3 WT1多表位基因疫苗的免疫原性研究3.1免疫指标检测免疫小鼠后实验结果显示:①特异性T淋巴细胞增殖实验:pcDNA-WT1免疫组和pcDNA-WT1- HSP70_407-426免疫组刺激指数同PBS、空质粒和pcDNA- HSP70_407-426各对照组相比显著升高（P<0.01）,但两组间无明显差异（P>0.05）;②CD4+/CD8+T淋巴细胞亚群:pcDNA-WT1免疫组和pcDNA-WT1-HSP70_407-426免疫组CD4+、CD8+T淋巴细胞数和CD4+/CD8+比值均显著高于PBS、空质粒和pcDNA- HSP70_407-426各对照组（p<0.01）,但两组之间无明显差异（p>0.05）;③WT1特异性的IFN-γ分泌频数:4条CTL表位肽混合物刺激各组脾脏淋巴细胞,ELISPOT检测结果显示,pcDNA-WT1免疫组和pcDNA-WT1-HSP70_407-426免疫组脾淋巴细胞中分泌IFN-γ的T细胞数较PBS、空质粒和pcDNA- HSP70_407-426各对照组显著增多（p<0.01）,但两组间无显著差异（p>0.05）;同时予4条CTL表位肽分别刺激pcDNA-WT1免疫组各小鼠脾脏淋巴细胞,均可检测到脾淋巴细胞特异性IFN-γ分泌,其中经WT1.235和WT1.126m多肽刺激组可检测到高水平的IFN-γ分泌,而经WT1.37或WT1.187多肽刺激组IFN-γ分泌水平较低,4组IFN-γ分泌水平均低于混合多肽刺激组（p<0.01）。④特异性杀伤实验显示:pcDNA-WT1免疫组和pcDNA-WT1- HSP70_407-426组免疫的CTL杀伤活性高于PBS对照组、空质粒组和pcDNA- HSP70_407-426组（p<0.01）,同样两组之间无明显差异（p>0.05）。3.2疫苗对肿瘤的治疗作用pcDNA-WT1免疫组和pcDNA-WT1-HSP70_407-426免疫组接种的FBL3肿瘤体积显著小于PBS对照组、空质粒对照组和pcDNA- HSP70_407-426对照组（p<0.01）,荷瘤小鼠生存期显著大于对照组（p<0.01）。3.3 HLA-A*0201限制性CTL反应pcDNA-WT1- HSP70_407-426转染的PBMC能有效诱导自身淋巴细胞产生CTL,细胞杀伤实验显示其能特异杀伤高表达WT1的肿瘤细胞并具有HLA-A*0201限制性。结论1在收集WT1表位的基础上,采用理论结合软件模拟的方法成功构建了WT1表位盒。2成功构建了WT1多表位基因疫苗pcDNA-WT1和融合基因疫苗pcDNA-WT1- HSP70_407-426。3构建的WT1多表位基因疫苗pcDNA-WT1能够有效诱导小鼠特异性免疫应答。4多表位基因疫苗pcDNA-WT1在小鼠体内具有抗瘤效应。5以mHSP70_407-426作为免疫佐剂构建的融合基因疫苗pcDNA-WT1- HSP70_407-426,与基因疫苗pcDNA-WT1相比,诱导的免疫应答及抑瘤效应无明显差别。6 WT1各CTL表位可以在多表位的模式下发挥各自功能。7 pcDNA-WT1-HSP70_407-426转染HLA-A*0201+PBMC能有效诱导自身淋巴细胞产生CTL,该CTL能特异杀伤高表达WT1抗原的肿瘤细胞,并受HLA限制性。更多还原

【Abstract】 ObjectiveThe WT1 gene was isolated as the gene responsible for a childhood renal reoplasm. In healthy adults , WT1 expression is limited to a set of tissues. However, the majority of acute leukemias and many solid tumors is ove rexpress WT1. And the xpression levels is an adverse prognostic factor in these malignant disease. The finding that blockade of WT1 function by WT1-antisense oligomers in primary leukemic cells significantly slows cell growth in vitro additionally suggests that WT1 is of critical importance to the tumor phenotype and that tumor escape by simple WT1 down-modulation or loss is unlikely to occur. These features of WT1 in terms of its biological functions and expression patterns indicate that WT1 could be an ideal cancer antigens for therapeutic vaccination.Up to date, WT1 antigen has been studied in clinical trials , and has showed a certain curative effect. However the efficiency of clinical trials is not satisfied because most of researchers use a single CTL epitope vaccination. The drawbacks inclued: inadequate activation of the innate immune system, T-cell epitope restriction to a particular MHC haplotype and possible immunoselection of epitope-loss variants.The WT1 polyepitope gene vaccines can make up the above drawbacks: （a）acompared to a single CTL epitope vaccine, the injection of multiple epitopes can overcome the potential loss of expression of a given -single epitope in cancer cells. （b） the vaccination with multiple epitopes would allow to enroll patients with different HLA moleular. （c） the vaccination with Th epitopes would not only elicit CTL responses, but also lyse the specific target cells. （d） The DNA vaccine can be easily design and construction.The gene adjuvant is an effective way to enhance genetic vaccine immunogenicity. It has been reported the adjuvant effect of mHSP70 is a effective adjuvant. The C-terminal stimulatory domain of mHSP70359-610 has the immuno adjuvant function, which can rapidly and directly induce the secretion of multiple cytokines from macrophages and dendritic cells by interacting with CD40. p407-426 is a stimulatory domain of HSP359-610. It could be more effectly induce DC mature than HSP359-610 , but with a better performance by reducing the self-immune responses caused by mHSP70. Therefore we chose the C-terminal stimulatory domain of mtHSP70.Based on the principles mentioned above, we constructed a WT1 polyepitope gene vaccines of which parts encode multiple CTL and Th epitopes, then we got the fusion genetic vaccine consisted of the mHSP70_407-426 and WT1 polyepitope gene. which was used to verify whether it has the activity to induce the immune responses and inhibit the growth of WT1-expressing tumor. Methods1. Design and synthesis of WT1 polyepitope cassettes Based on collecting published WT1 epitopes, WT1 cassettes of good immunogenicity with CTL and Th epitopes were designed. Then the cassettes were optimized by computer-based modeling . Ulitimately, the cassettes were synthesized.2. Design and construction of WT1 polyepitope genetic vaccine2.1 Construction of eukaryotic expression plasmidThe synthetic nucleotide sequence was cloned into pUC57-T easy vector. After digestion, it was cloned the eukaryotic vector to construct the plasmid pcDNA-WT1. For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplificated by polymerase chain reaction from pGEM-mHSP70 vector and cloned into pcDNA3.1（+）. Then WT1 polyepitopewas fused to the N-terminal of pcDNA-mHSP70_407-426 to make the multi-epitope fusion gene vaccine pcDNA-WT1-HSP70_407-426.2.2 Transfecion of genetic vaccine to 293T /Hela cellsHela and 293T cells were transfected with empty pcDNA、pcDNA- HSP70_407-426、pcDNA-WT1 and pcDNA-WT1-HSP70_407-426 vector by liposome transfection.2.3 RT-PCR and western blotTransfect HEK-293T cells with genetic vaccine and identify the expressed product by RT-PCR. Transfect Hela cells with the constrcuted DNA vaccine pcDNA-WT1-HSP407-426 and identify the expressed product by Western blot.3 Study of immunogenicity of WT1 polyepitope genetic vaccine3.1 Vaccination of miceThe immunogenicity of the vaccine was assessed using male 4-6 week-old C57BL/6mice, divided into 5 groups. The mice were immunized with the pcDNA-WT1 and with pcDNA-WT1-HSP70_407-426, pcDNA-HSP70_407-426, empty pcDNA vector and PBS as controls. Each mouse was inoculated into each tibialis muscle with with 0.25 % bupivacaine and then after 48 hour with 50ug plasmid every 2 weeks intramuscularly for three times. After 10 days of the last time of inoculation, we isolated the lymphocytes of the mice’s spleen.3.2 The immunogenicity of the genetic vaccineT lymphocyte proliferation by MTT, T lymphocyte subpopulations by FCM, peptide-specific T cells by ELISPOT assay and cytotoxicity by LDH release assay evaluated the genetic vaccine immunity resposes.3.3 Effect of genetic vaccine on therapy of established tumorsMale C57BL/6 mice, 4 to 6 weeks old, were selected and divided into 5 groups. Before the start of the treatments , five groups mice were inoculated with 2×106 FBL3 tumor cells. At the 7 days after the inculation, mice were immunized with the pcDNA-WT1, with pcDNA-WT1-HSP70_407-426, pcDNA- HSP70_407-426, empty pcDNA vector and PBS as controls. The muscle DNA administrations were repeated at 14 days after the first treatment, and during this time the tumor sizes in the mice were recorded every five days.3.4 Induction of HLA-A*0201-restricted CTLPBMC were isolated from the blood of HLA-A*0201+ healthy donors .The fusion genetic vaccine pcDNA-WT1-HSP70_407-426 were transfected into PBMC to stimulate autologous lymphocytes in vitro. The effector fuction of inudced CTL were analyzed in cytotoxicity assays.Results1 Design and synthesis of WT1 polyepitope cassettes1.1 Screening of WT1 epitopesThe selection WT1 epitopes comply with two basic principle. First it has advantage in induction of CTL or Th immune response. Second it can bind to more MHC molec-ular. So the selected CTL epitopes included WT1.37（VLDFAPPGA;HLA*0201）、WT1.187（SLGEQQYSV;HLA*0201/HLA*0206） and WT1.235（CMTWNQMNL; HLA*0201/HLA*2402）; Th epitopes included WT1.121A（SGQAYMFPNAPYLPSCLES;HLA-DRB1*0401） and WT1.332（KRYFKLSHLQMHSRKH,HLA-DRB1*0405/HLA-DRB1*1501/HLA-DRB1*1502/HLA-DPB1*0901）.And the WT1.122A concealed a CTL epitoes WT1.126（underline,HLA-A*0201/H2Db）. In order to break through the MHC restriction better,one universal Th Pan-DR epitope （PADRE, KFVAAWTLKAAA） was also selected.1.2 Design and optimization of the cassettesIn order to make every epitope has its independent function, the portion of the gene encoding CTL epitopes were designed using computer-based modeling（PAPro and NetChop ）to optimize proteasome mediated epitope processing through the introduction of amino acid spacer sequences, such as AAY、KK、KAA、KAAA、AAA and NAAA. The portion of the gene encoding the HTL epitopes was designed with GPGPY amino acid spacers between sequential HTL epitopes . The IgGκchain leader sequence was used as signal peptide, and inserted Kozak sequence at the 5’-terminal of signal peptide as the ribosome-binding site. Codon optimization of nucleotide sequence by DyNAVacS. Ultimately the cassettes by software proved that the design was consistent with that expected in theorpy .2. Design and construction of WT1 polyepitope genetic vaccine2.1 Construction of eukaryotic expression plasmidRestriction endonuclease digestion analysis and PCR result of the recombinant vector pcDNA-WT1、pcDNA -HSP70_407-426 and pcDNA-WT1-HSP70_407-426 showed that were indentical with aim gene fragments, and DNA sequence analysis showed WT1 and HSP70 was right in the recombinant plasmid . It is concluded that all vector were successfully designed.2.2 Transfecion of genetic vaccine to 293T /Hela cellsCompared with non-tracsfected cell, EGFP expression was only observed in cotrans -fected cells 293T/Hela that showed pcDNA-WT1、pcDNA-HSP70_407-426 and pcDNA-WT1 -HSP70_407-426 could be expressed in eukaryotics.2.3 RT-PCR and western blotAfter 48h of being transfected with 293T and Hela cell, RT-PCR showed correct expression of target gene in HEK-293T cells and western blot showed correct expression of WT1 protein in Hela cells by pcDNA-WT1-HSP70_407-426 transfection.3 Study of immunogenicity of WT1 polyepitope genetic vaccine3.1 The immunogenicity of the genetic vaccineThe result of T lymphocyte proliferation by MTT indicated that immunized mice by pcDNA-WT1-HSP70_407-426 and pcDNA-WT1-HSP70_407-426 had significantly higher levels of stimulation index compare to PBS、pcDNA and pcDNA-HSP70_407-426 controls（p<0.01）. Flow cytometry results showed that immunized mice by pcDNA-WT1 and pcDNA-WT1-HSP70_407-426 had significantly higher levels of CD4+/CD8+ compare to three controls（p<0.01）. The immunized mice also have significantly more T cells and higher CD4+/CD8+ values than controls（p<0.01）.The result of IFN-γdetection by ELISOPT indicated that there are more numbers of IFN-γ-secreting CD8+T cells induced by peptide mixtures in pcDNA-WT1 and pcDNA-WT1-HSP70_407-426 than that of three controls（p<0.01）. Furthermore, pcDNA-WT1 immunized mice induced by four CTL peptides individually, all of them can detect specific IFN-γ-secreting. LDH release assay experiments showed that the immunized mice had higher CTL killing-effects than three controls. While according to above-mentioned experiment, there was no significant different between pcDNA-WT1 and pcDNA-WT1-HSP70_407-426 group（p>0.01）. Comparing with PBS, pcDNA and pcDNA-HSP70_407-426 group, the frequency of WT1^-specific CTL was more and cytotoxicity to FBL3 cells were higher in pcDNA-WT1 and pcDNA-WT1^-HSP70_407-426 group（p<0.01）. However, there had no significant difference between pcDNA-WT1 and pcDNA-WT1^-HSP70_407-426（p>0.01）, that indicate pcDNA-WT1^-HSP70_407-426 can not enhance the potential of pcDNA-WT1 vaccine.3.3 Effect of genetic vaccine on therapy of established tumorsThe tumor volume was significantly lower in the pcDNA-WT1 and pcDNA-WT1^-HSP70_407-426 than three controls（p<0.01）.And the survival time was longer in the pcDNA-WT1 and pcDNA-WT1^-HSP70_407-426 than controls（p<0.01）.3.4 Induction of HLA-A*0201-restricted CTLThe human pcDNA-WT1^-HSP70_407-426 transfected PBMC through making HLA-A*0201 restrticted CTL assay. CTLs could recognize and lyse NB4（HLA-A*0201+WT1⁺）. Whereas control target U937（HLA-A*0201+WT1^-）, or K562(HLA-A*0201-WT1⁺ were not killed by the CTLs. it appears that tumour cell lysis was WT1 specific and HLA-A0201-restricted.Conclusions1 The expression cassette for multiple-epitope antigen of WT1 was successfully designed.2 The genetic vaccine pcDNA-WT1 and fusion vaccines pcDNA-WT1^-HSP70_407-426 were successfully constructed.3 The WT1 polyepitopegenetic vaccines could elicit WT1^-specsific cellular immunity and have therapeutic effects against the WT1^-expressing tumors.4 The WT1 poly-epitope genetic vaccines had potential therapeutic effects against WT1^-expressing tumors.5 The linkage of mHSP70_407-426 to WT1 can not enhance the potengcy of WT1 vaccines.6 Every CTL epitopes could have its independent function.7 The pcDNA-WT1^-HSP70_407-426-transfected HLA-A0201+ PBMC are remarkably effective in stimulating potent HLA-A*0201-restricted CTL.更多还原