CEA 迷你基因串联体疫苗免疫小鼠脾细胞对CEA 阳性肿瘤细胞的杀伤作用及疫苗的安全性评价

目的:观察CEA 迷你基因串联体疫苗pcDNA-triCEA625-667 免疫小鼠脾细胞对肿瘤细胞特异性杀伤作用并对疫苗免疫小鼠后的安全性进行评估。方法: BALB/ c 小鼠随机分为空白载体组(pcDNA3.0)、单倍体疫苗实验组(pcDNA-CEA625-667 )、串联体疫苗实验组(pcDNA-triCEA625-667 ),肌肉注射法免疫动物,每隔10 d 免疫1 次,共免疫4 次,记录免疫小鼠的体重变化、存活情况以及检测血清ALT、肌酐水平。以疫苗免疫小鼠的脾细胞为效应细胞,以LDH 释放法检测其对CEA 阳性的小鼠肝癌细胞株(H22-CEA+ )、胃癌细胞株(MFC-CEA+ )、结肠癌细胞株(CT26-CEA+ )以及CEA 阴性小鼠肝癌细胞株(H22-CEA- )的特异性CTL 的杀伤活性。结果:两种疫苗对CEA 阳性的肝癌、胃癌及结肠癌细胞均具有较强的杀伤活性,与PcDNA3.0 空载体组相比,差异有统计学意义(P<0.01)。而对CEA 阴性肿瘤细胞(H22-CEA- )则几乎无影响。迷你串联体基因疫苗pcDNA-triCEA625-667 对肝癌细胞H22-CEA+及胃癌细胞MFC-CEA+的杀伤活性强于单倍体基因疫苗pcDNA-CEA625-667(P<0.05)。疫苗免疫对小鼠的存活状态、体重变化及肝肾功能指标均无影响。结论:CEA 迷你基因疫苗安全有效,能够诱导肿瘤特异性CTL 产生,且三倍体串联体疫苗免疫效果优于单倍体疫苗。     

CEA 迷你基因串联体疫苗的体内抗肿瘤活性观察

目的:观察已构建的含有CEA625-667 基因单倍体疫苗pcDNA-CEA625-667 及三串联体的DNA 疫苗pcDNA-triCEA625-667 对荷瘤小鼠体内肿瘤的抑制情况及小鼠存活时间的改变。方法:建立小鼠肝细胞癌实验动物模型,应用单倍体疫苗pcDNA-CEA625-667 及三串联体DNA 疫苗pcDNA-triCEA625-667 免疫小鼠,以生理盐水为对照组,观察各实验组小鼠皮下肿瘤生长情况,记录皮下肿瘤生长曲线,观察疫苗对荷瘤小鼠肿瘤生长速度的影响以及疫苗对荷瘤小鼠生存时间的影响。结果:与生理盐水对照组相比,两种疫苗均能明显抑制CEA 阳性荷瘤小鼠的肿瘤体积以及生长速度(P <0.01),其中,pcDNA-triCEA625-667 疫苗组的抑制作用明显优于pcDNA-CEA625-667 疫苗组(P<0.01),而二者均不能抑制CEA 阴性荷瘤小鼠的肿瘤生长。pcDNA-CEA625-667 疫苗组平均生存时间为(48.50±6.73)d,与生理盐水对照组(39.00±6.64)d 相比有显著差异(P<0.01);pcDNA-triCEA625-667 疫苗组生存时间(48.50依6.73)d 明显高于生理盐水对照组和pcDNA-CEA625-667 疫苗组(P<0.01)。两组疫苗均不能延长CEA 阴性荷瘤小鼠的生存时间。结论:无论是单倍体还是三串联体的DNA 疫苗,均能够明显抑制CEA 阳性荷瘤小鼠的肿瘤生长速度(P<0.01)及明显延长其生存时间(P<0.01),而对CEA 阴性荷瘤小鼠则无治疗作用。     

CEA 迷你肽表位基因疫苗诱导小鼠抗肿瘤免疫的研究

目的:利用含有CEA625-667 基因的单倍体疫苗pcDNA-CEA625-667 及三串联体的DNA 疫苗pcDNA-triCEA625-667 免疫小鼠后,观察其诱导的抗肿瘤免疫效应。方法: 采用4 ~ 6 周纯系BALB/ c 小鼠,肌肉注射法分pc-DNA3.0、pcDNA-CEA625-667 、pcDNA-triCEA625-667 三组免疫小鼠,对其激发的机体特异性及非特异性免疫反应进行研究。流式细胞术检测免疫小鼠脾细胞的T 细胞亚群和CD4+ / CD8+比值;3 H-TdR 掺入法检测免疫小鼠的特异性淋巴细胞增殖;Western blot 杂交及ELISA法检测免疫小鼠血清中的CEA 特异性抗体;ELISA 法检测免疫动物脾细胞体外诱导IFN-β、IL-4、GM-CSF 的分泌水平。结果:实验组与对照组的CD4+ / CD8+比值差异无统计学意义;经过基因疫苗免疫的小鼠与天然小鼠相比,其脾细胞在体外与短肽共孵育之后,会在更短的时间内出现更明显的细胞增殖;免疫了CEA 迷你基因串联体肿瘤疫苗的小鼠血清中可以产生低滴度的抗体,提示HTL 的活化;免疫小鼠脾细胞上清中IFN鄄酌的含量明显高于对照组,而pc-DNA3.0,pcDNA-CEA625-667 ,pcDNA-tri-CEA625-667各组IL-4 的含量均很低,差异无统计学意义;迷你基因三倍体疫苗所引发的增殖效应以及释放细胞因子的水平均高于迷你基因一倍体,说明我们所采用将目的基因多倍串联的抗原改造的方式起到了增强免疫效应的作用。结论:CEA 迷你肽表位基因单倍体及三倍体疫苗均不能显著改变动物体内CD4/ CD8 比值,但均能诱导HTL 活化,使被免疫机体T 细胞趋向于Th1 效应且三倍体疫苗的免疫原性强于单倍体疫苗。     

癌胚抗原基因重组疫苗的构建及免疫效果观察

目的：探讨含癌胚抗原（CEA）部分编码基因的真核重组质粒pIRES-CEAⅢ用于CEA阳性肿瘤免疫治疗可能性。方法：应用基因重组技术构建了含CEA信号肽和Ⅲ区编码序列的真核表达质粒pIRES，CEAⅢ，肌注免疫BALB／c小鼠，再分别应用野生型小鼠肝癌细胞H22及CEAcDNA全序列转染的Hn细胞进行小鼠皮下接种，观察基因重组疫苗对CEA阳性肿瘤的抑制作用及其诱导小鼠细胞毒性T淋巴细胞（CTL）特异性杀伤CEA阳性肿瘤细胞的能力。结果：pIRES-CEAⅢ真核重组质粒免疫组小鼠CEA阳性肿瘤的生长速度趋缓，瘤块较小，与对照组小鼠（免疫空载体质粒和接种野生型H控细胞）相比有明显差异（P〈0．05）；经pIRES-CEAⅢ重组质粒免疫的小鼠脾细胞对H22．CEA（＋）细胞的杀伤率明显升高，差异显著（P〈0．01）；但对H22细胞则没有明显的杀伤作用。结论：pIRES-CEAⅢ作为基因疫苗可以抑制CEA阳性肿瘤在小鼠体内的生长，诱导小鼠CTL对CEA阳性肿瘤细胞的特异性杀伤。     

探讨BCG初次免疫结核杆菌DNA疫苗加强免疫方案的效应

目的:探讨BCG初次免疫(BCG-prime),结核杆菌共表达DNA疫苗加强免疫(DNA疫苗-boost)的策略对小鼠的免疫效果。方法:将BCG及结核杆菌重组DNA疫苗依次免疫小鼠,通过检测CTL和NK细胞的杀伤活性和特异性淋巴细胞增殖,以及小鼠血清抗体及细胞因子的水平,观测BCG-prime、共表达结核杆菌Ag85A/GM-CSFDNA疫苗boost策略对小鼠的免疫效果。结果:采用prime-boost免疫策略组的小鼠CTL的杀伤活性明显增强、特异性淋巴细胞明显增殖、IFN-γ的水平明显增高,NK细胞杀伤活性与对照组相比也有一定提高,但未超过BCG单独免疫效果。免疫小鼠血清特异性抗体的滴度超过单独DNA疫苗免疫组。结论:在采用BCG-prime-结核杆菌DNA疫苗boost免疫策略后,能增强对小鼠的免疫效应,尤其是Th1型细胞免疫反应增强明显,为进一步在动物体内进行保护性效应试验的研究提供了实验依据。     

共表达HIV-1 gp120与IFN-α重组鸡痘病毒诱导特异性CTL的初步研究

目的:探讨共表达HIV -1gp12 0与IFN- α重组鸡痘病毒诱导小鼠产生特异性的CTL杀伤活性。方法:将重组鸡痘病毒经肌肉注射免疫BALB c小鼠后,制备小鼠脾淋巴细胞悬液。以免疫小鼠的脾淋巴细胞为效应细胞,以表达HIV- 1结构蛋白的P815细胞为靶细胞,用乳酸脱氢酶释放法测定免疫小鼠脾特异性CTL杀伤活性。结果:重组病毒可有效地诱导特异性CTL的产生,且重组病毒免疫组小鼠脾特异性CTL对靶细胞的杀伤活性显著高于FPV对照组(P <0 .0 1)和PBS对照组(P <0. 0 1)。结论:共表达HIV- 1gp12 0和IFN -α的重组鸡痘病毒可激发强烈的细胞免疫,可作为我国HIV- 1疫苗候选株。     

新城疫病毒HN基因对肿瘤抗原诱导的抗肿瘤免疫的增强作用

目的 了解新城疫病毒HN基因增强肿瘤抗原诱发的抗肿瘤免疫反应的作用。并对其作用机制进行探讨。方法 将构建的HN基因-癌胚抗原（CEA）cDNA共表达质粒（pcD-CEA/HN)免疫小鼠,通过淋巴细胞增殖实验,NK细胞活性检测了解HN对抗CEA免疫反应的影响,并以免疫组化的手段追踪HN质粒表达产物在体内组织的分布,以及HN质粒对荷瘤小鼠肿瘤生长,抗肿瘤免疫反应的影响。结果 （1）共表达质粒pcD-CEA/HN免疫小鼠获得了较其他对照组强的淋巴细胞增殖指数及NK细胞活性。（2）HN质粒在肌肉,肿瘤组织有明显的表达。（3）HN质粒对肿瘤长生具有一定的抑制作用。并能增强荷瘤小鼠的抗肿瘤免疫。结论 新城疫病毒HN基因对肿瘤抗原诱导的抗肿瘤免疫具有增强作用。     

对携带HCC表位的HBc病毒样颗粒负载的DC疫苗的免疫评价

目的:探讨携带肝细胞癌(HCC)表位的嵌合蛋白颗粒HBc-VLPs负载小鼠BMDCs制备的DC疫苗,免疫小鼠后诱导抗原特异的细胞免疫应答和抗肿瘤效应。方法:制备HLA-A2转基因小鼠来源的BMDCs,将三种带有高表达于HCC的HLA-A2限制的CTL表位的HBc-VLPs、三种抗原表位肽和不含HCC表位野生HBc-VLPs分别负载HLA-A2转基因小鼠BMDCs制备成DC疫苗,阴性对照组用PBS替代。将HLA-A2转基因小鼠分为四组,分别免疫上述DC疫苗并对其免疫效果进行评价:体外检测抗原特异性淋巴细胞内IFN-γ的产生和CTL细胞活性并观察不同方法处理的DCs对肿瘤生长的抑制情况。结果:与其他组相比,HBc-VLPs负载的DCs免疫小鼠后第8天,流式细胞仪即可检测到分泌IFN-γ的CD8+T细胞存在;用LDH法检测到较强的抗原特异性CTL活性;在肿瘤接种后的20天之前均未见到明显的肿瘤块,具有显著抗肿瘤作用。结论:利用携带HCC抗原表位的HBc-VLPs负载DCs后得到可强烈诱导免疫活性和明显抗肿瘤作用的DC疫苗,为DC疫苗的优化和评价提供了实验方法,也为进一步深入研究新型肝细胞癌治疗性疫苗的功能和应用奠定了基础。     

CEA迷你基因原核表达及其诱导小鼠特异性淋巴细胞增殖的研究

目的 在原核细胞中表达人癌胚抗原(CEA)的迷你基因短肽,纯化及鉴定目的 蛋白,并检测其在小鼠体内的抗原性.方法 利用PCR技术从人基因组中得到一段来源于CEA的DNA序列,其中包含两个编码分别能被HLA-DR4/9、HLA-DR53以及HLA-DR4/7/9型别的人群所识别辅助性T细胞(HTL)表位的迷你基因,构建重组表达质粒pQE30-CEA625-667,在大肠杆菌M15中诱导表达.Westren Blot杂交鉴定、镍凝胶亲和层析法纯化目的 蛋白.3H-TdR掺入法检测目的 短肽诱导的小鼠淋巴细胞增殖反应.结果 CEA625-667短肽在大肠杆菌M15中以包涵体形式表达.Western-blot结果显示,在相对分子质量约6 700处有表达产物与6×his mAb特异性结合带.镍柱纯化后可得到纯化的目的 蛋白.3H-TdR掺入实验所得不同浓度的CEA625-667与小鼠脾细胞共孵育7～9 d后的刺激指数相继达到对照组的10倍以上.结论 成功诱导了CEA625-667短肽的原核表达,通过镍凝胶亲和层析法获得纯度较高的CEA625-667短肽,并证明在一定浓度下目的 短肽可于体外诱导小鼠的特异性淋巴细胞增殖.为CEA625-667迷你基因作为表位疫苗在抗肿瘤方面的进一步研究提供条件.     

点突变p53及其抗原肽重组痘苗病毒抗肿瘤效应的比较研究

目的　比较点突变p5 3及其抗原肽重组痘苗病毒诱导的抗瘤免疫反应 ,为重组抗原疫苗用于肿瘤免疫治疗提供实验依据。方法　以人 135位Cys→Tyr点突变 p5 3为肿瘤相关抗原模型 ,观察以表达该点突变 p5 3蛋白的重组痘苗病毒rVV p5 3FL与表达包含该点突变抗原肽 p5 312 5 14 5的重组痘苗病毒rVV p5 3M 所诱导的CTL及对荷瘤Balb/c小鼠的免疫保护和治疗作用。结果　rVV p5 3FL和rVV p5 3M 均能诱导以CD8 T细胞为主的特异性CTL。用rVV p5 3FL与rVV p5 3M 免疫小鼠后 ,接种致死剂量的P815 mp5 3细胞 ,能保护部分小鼠免遭致死剂量肿瘤细胞的攻击。小鼠接种致死剂量的肿瘤细胞后 ,以rVV p5 3FL与rVV p5 3M 治疗荷瘤小鼠 ,可使小鼠平均存活时间显著延长。两种疫苗的抗瘤效应无明显区别。结论　来源于突变p5 3的抗原肽重组疫苗 ,可代替抗原大分子应用于肿瘤的免疫防治     

小鼠骨髓树突状细胞pcDNA3-hCEA转染疫苗的抗瘤作用

目的:将pcDNA3-hCEA转染小鼠骨髓树突状细胞 (DCs),观察其诱导BALB/c小鼠对CT2 6(hCEA⁺)的抗肿瘤免疫效应。方法:采用rmGM-CSF和rmIL-4体外诱导培养小鼠骨髓DCs;构建真核表达质粒pcDNA3-hCEA;并用lipofectamine将其转染DCs,制备DCs(pCDNA3-hCEA)疫苗;同时制备CT2 6(hCEA⁺);采用RT-PCR检测DCs(pCDNA3-hCEA)中CEAmRNA的表达,采用放射免疫法检测DCs(pCDNA3-hCEA)培养上清中CEA的含量;使用DCs(pCDNA3-hCEA)疫苗体外诱导小鼠同种异体T淋巴细胞靶向性杀伤。采用DCs(pcDNA3-hCEA)疫苗预防免疫BALB/c小鼠,观察其对于荷临界致瘤量CT2 6(hCEA⁺)小鼠的抗肿瘤效应。结果:经G₄₁₈筛选,DCs(pCDNA3-hCEA)有14 %的获得率;RT-PCR检测表明DCs(pCDNA3-hCEA)内CEAmRNA呈阳性表达;放射免疫法检测表明DCs(pCDNA3-hCEA)能微量表达人CEA;DCs(pCDNA3-hCEA)能够有效诱导CEA靶向免疫杀伤。DCs(pcDNA3-hCEA)疫苗延长荷CT2 6(hCEA⁺)瘤小鼠生存期1周至 4周。结论:编码肿瘤抗原基因的真核表达质粒转染的树突状细胞,能够诱导较强的特异性抗肿瘤免疫效应.     

Pre-clinical evaluation of a CEA DNA prime/protein boost vaccination strategy against colorectal cancer

In preparation for a clinical trial in patients diagnosed with colorectal cancer, a vaccination strategy targeting the carcinoembryonic antigen (CEA) was evaluated in mice using a GMP-produced plasmid DNA vaccine, CEA66, encoding a truncated form of the tumour-associated antigen, CEA. The GMP-produced CEA DNA vaccine was also evaluated for toxicity. Repeated intradermal administration of the GMP-produced vaccine using a novel needle-free jet injection device (Biojector) induced robust CD4 and CD8 T-cell responses in mice, and did not result in any vaccine-related toxicity. In a heterologous DNA prime/protein boost setting, cellular immune responses were of higher magnitude in animals primed with CEA66 DNA than in animals receiving repeated doses of recombinant CEA protein. These responses were further enhanced if recombinant murine granulocyte-macrophage colony-stimulating factor was given as an adjuvant prior to vaccination. In contrast to repeated administration of recombinant CEA protein as a single modality vaccine, the heterologous CEA66 DNA prime/rCEA boost vaccination strategy resulted in a qualitatively broader immune response, and supports clinical testing of this vaccination regimen in humans.     

A novel recombinant DNA vaccine encoding Mycobacterium tuberculosis ESAT-6 and FL protects against Mycobacterium tuberculosis challenge in mice

Mycobacterium tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand (FL) that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid (pIRES-epitope-peptides-FL) encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine (prime) and intranasal administration of the epitope peptides (boost) was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Th1 cytokines (IFN-γ and IL-12), the number of IFN-γ⁺ T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant pIRES-epitope-peptides-FL plasmid is a useful DNA vaccine for preventing Mycobacterium tuberculosis infection.     

DNA vaccine expressing repeated carcinoembryonic antigen (CEA)(625-667) induces strong immunity in mice

The efficacy of immunization with DNA plasmids for single truncated carcinoembryonic antigen (CEA) peptide or triple repeated CEA peptides in mice was evaluated. A DNA fragment the truncated CEA gene (nucleotide 625-667) encoding two helper T lymphocyte (HTL) epitopes was amplified by PCR and cloned for generating recombinant plasmids for single CEA(625-667) (pcDNA-CEA(625-667)) or triple CEA(625-667) (pcDNA-triCEA(625-667)), respectively. Subsequently, groups of BALB/c female mice were intramuscularly injected with pcDNA-CEA(625-667,) pcDNA-triCEA(625-667) or control pcDNA3.0 vector, respectively. Ten days after the last immunization, the frequency of splenic CD4(+) and CD8(+) T cells in the mice was determined by flow cytometry. The antigen-specific proliferation of splenic T cells and cytokine production ex vivo were analyzed by (3)H-TdR uptake and cytokine ELISA, respectively. The levels of serum antibodies against CEA in the mice were detected by Western blot and ELISA. Although immunization with plasmid for the CEA(625-667) peptide(s) did not alter the frequency of CD4(+) and CD8(+) T cells in mice, vaccination with plasmid for CEA peptide induced strong antigen-specific T cell proliferation, particularly for the plasmid encoding the triple-repeated CEA peptides, accompanied by significantly elevated levels of IFN-γ secreted by T cells from the mice immunized with triple-repeated peptides. Furthermore, immunization with the plasmid for CEA peptide stimulated higher levels of antigen-specific antibody responses in mice. Vaccination with the plasmid for the triple repeated CEA peptides induced stronger Th1 responses. Our findings may be useful for the development of effective DNA vaccine for the immunotherapy of cancer.     

Induction of immune response and anti-tumor activities in mice with a DNA vaccine encoding human mucin 1 variable-number tandem repeats

Mucin 1 (MUC1) is a tumor-associated antigen that carries the important variable-number tandem repeat (VNTR) epitopes for inducing cytotoxic T lymphocytes. Such a property makes MUC1 VNTR potentially attractive for immunotherapy. This study explored the possibility of developing an efficient anti-tumor vaccine strategy using the specific antitumor immunity induced by the MUC1 VNTR DNA vaccine combined with the adjuvant effect of a plasmid expressing murine interleukin-2 (IL-2). The results showed that the MUC1 VNTR DNA vaccine successfully induced both humoral and cellular immune responses against MUC1 VNTR in mice. The effect could be obviously enhanced by increasing the number of tandem repeats, the number of immunizations, and by co-administration of the cytokine plasmid. The growth of MUC1-expressing (MUC1(+)) tumors was significantly inhibited in mice immunized with the MUC1 VNTR DNA vaccine combined with the IL-2 plasmid, both before and after tumor challenge. A much larger percentage of the immunized mice survived tumor challenge than the non-immunized mice. The combination of the MUC1 VNTR DNA vaccine and the IL-2 adjuvant plasmid provides an attractive alternative for prophylactic and therapeutic vaccinations against MUC1(+) tumors.     

Intramuscular co-injection of naked DNA encoding HBV core antigen and Flt3 ligand suppresses anti-HBc antibody response

Flt3 ligand, a recently described growth factor affecting early hematopoietic progenitor cells, can also support the expansion of dendritic cells secreting IL-12. Its potential use in a clinical setting has been suggested. Here, we studied the effect of in situ delivery of Flt3 ligand plasmid (FL) on the antibody response induced by DNA vaccine encoding wild-type hepatitis B virus core antigen (HBc/w). Intramuscular injection of FL increased the expression of DEC205 and the size of splenocytes, and immunization with HBc/w or HBc/w-transfected EL-4 cells induced strong anti-HBc antibody responses in mice. However, intramuscular injection of FL with HBc/w significantly suppressed HBc/w-induced antibody response in a dose-dependent manner. Suppression of immune response by FL injection was the most prominent when FL and HBc/w were co-injected at the same time and the same site. These results suggest that FL may inhibit humoral response induced by DNA-type vaccination, and DC locally expanded by FL may not have proper functions for induction of humoral response.     

PSMA基因疫苗抑瘤效应及免疫机制的实验研究

目的： 观察表达前列腺特异性膜抗原(PSMA)基因疫苗在荷瘤小鼠模型中抑瘤效应并探讨其免疫机制,为前列腺癌的预防和免疫治疗提供实验基础。方法: 将PSMA基因疫苗肌注入BALB/c小鼠体内,检测其血清中PSMA抗体水平并观察脾T细胞的增殖效应和杀细胞毒效应,以sp2/0-PSMA细胞攻击免疫后小鼠,观察小鼠的成瘤率、肿瘤大小、平均瘤重及生存率,评价PSMA基因疫苗的抑瘤效应。结果: PSMA基因疫苗可诱导实验组小鼠产生PSMA抗体,脾T细胞的增殖效应和杀细胞毒效应,在一定时间内随着免疫次数的增加和时间的延长,抗体水平、脾细胞的增殖和杀细胞毒效应均呈上升趋势。与对照组相比较,实验组小鼠成瘤率低,无瘤生存期延长,肿瘤生长缓慢。结论: PSMA基因疫苗能诱导实验组小鼠产生特异性体液及细胞免疫应答,且有明显的抑瘤效应。