DNA疫苗与抗肿瘤免疫

DNA疫苗是近年发展起来的新一代疫苗，能够诱导宿主体内细胞毒T细胞反应和抗体反应。本量实验研究证明，注射抗肿瘤DNA疫苗能获得肿瘤保护性免疫效应，在肿瘤免疫治疗中应用前景看好。本文对DNA疫苗抗肿瘤免疫的机制，影响因素及应用作一综述。     

DNA疫苗与抗肿瘤免疫

DNA疫苗是近年发展起来的新一代疫苗 ,能够诱导宿主体内细胞毒T细胞反应和抗体反应。大量实验研究证明 ,注射抗肿瘤DNA疫苗能获得肿瘤保护性免疫效应 ,在肿瘤免疫治疗中应用前景看好。本文对DNA疫苗抗肿瘤免疫的机制、影响因素及应用作一综述     

006 DNA疫苗与抗肿瘤免疫

DNA疫苗是近年发展起来的新一代疫苗,能够诱导宿主体内细胞毒T细胞反应和抗体反应.大量实验研究证明,注射抗肿瘤DNA疫苗能获得肿瘤保护性免疫效应,在肿瘤免疫治疗中应用前景看好.本文对DNA疫苗抗肿瘤免疫的机制、影响因素及应用作一综述.     

DNA疫苗用于癌症的治疗

近来发现注入编码各种蛋白的质粒可在体内产生相应的ＣＴＬ和抗体，使接种质粒成为一种新的特异性免疫方法，它可引起ＭＨＣ－Ⅰ和ＭＨＣ－Ⅱ介导的免疫应答，使之可能成为发展肿瘤疫苗的理想方法。根据癌变的遗传学，用质粒可有效地构建亚单位和多亚单位疫苗，ＤＮＡ编码的种瘤特异性蛋白质片断作为新抗原可打破免疫     

树突状细胞靶向性DNA疫苗的抗肿瘤免疫效应

目的:构建含OVA-Fc融合基因并对树突状细胞具有靶向性的DNA疫苗,评价其在肿瘤治疗中的作用。方法:构建真核表达载体OVA-Fc-pcDNA3.1,以脂质体转染法将其导入CHO细胞,用流式细胞术和ELISA法检测融合蛋白OVA-Fc的表达。建立E.G7-OVA荷瘤小鼠模型,用51Cr释放实验测定免疫小鼠脾脏细胞毒性T淋巴细胞(CTL)的抗肿瘤活性。通过观察荷瘤小鼠肿瘤的体积和生存期评价该肿瘤疫苗的疗效。结果:酶切鉴定和序列测定证明,真核表达载体OVA-Fc-pcDNA3.1构建正确。用流式细胞术和ELISA法均表明,转染的CHO细胞能表达OVA-Fc融合蛋白。OVA-Fc能激发CTL的杀伤活性,发挥抗肿瘤作用,从而减缓肿瘤的生长,延长荷瘤小鼠的生存期。结论:含OVA-Fc-pcDNA3.1的树突状细胞靶向性DNA疫苗能在体内有效地激发抗瘤免疫应答,为进一步开展临床实验奠定了基础。     

DNA疫苗抗肿瘤研究进展

质粒ＤＮＡ或称裸ＤＮＡ疫苗直接肌肉注射后,能表达编码基因的蛋白,并诱导宿主免疫反应。这为肿瘤基因－免疫治疗提供了一种新的途径。近１０年的研究表明,ＤＮＡ疫苗表达质粒骨架中的免疫刺激序列具有佐剂效应和促有丝分裂作用,这与免疫反应的诱导有密切关系。注射部位的肌细胞起着抗原蛋白表达储池作用,通过骨髓来源的抗原呈递细胞,最终诱发宿主体内ＣＴＬ和抗体反应。ＤＮＡ疫苗抗肿瘤的靶向性是由插入表达质粒的编码基因所     

pcDNA3.1+/MAGE-3 DNA疫苗诱导特异性抗肿瘤免疫应答的实验研究

目的：构建pcDNA3.1+/MAGE-3 DNA疫苗，观察其在小鼠体内诱导特异性抗肿瘤免疫应答的能力。方法： 通过RT-PCR构建重组表达质粒pcDNA3.1+/MAGE-3；以pcDNA3.1+/MAGE-3 DNA疫苗免疫已接种肿瘤细胞的小鼠，每10 d重复免疫1次，共3次，以pcDNA3.1+、PBS为对照。末次免疫后5 d检测血清中MAGE-3抗体滴度、小鼠脾淋巴细胞的细胞毒T细胞（cytotoxic T lymphocytes，CTL）杀伤活性、细胞因子IL-2和IFN-γ的浓度，同时计算抑瘤率。结果： 成功构建了pcDNA3.1+/MAGE-3 DNA疫苗，用此疫苗免疫已接种B16/MAGE-3细胞的小鼠后，能诱导小鼠脾淋巴细胞MAGE-3特异性的杀伤活性，脾细胞培养上清中细胞因子IL-2和IFN-γ的浓度明显增高，血清中抗MAGE-3抗体在1∶20滴度时阳性，肿瘤生长被显著抑制，与pcDNA3.1+组、PBS组相比，差异显著（P＜0.01）。结论： 成功构建了pcDNA3.1+/MAGE-3 DNA疫苗，该疫苗在小鼠体内既能激活CTL杀伤活性和CD4+ T细胞活性，又能激活体液免疫反应，从而诱导出特异性的抗肿瘤免疫应答。     

IL—6基因转染的瘤苗体内诱导的抗肿瘤免疫功能与效果

经丝裂霉素C体外处理后,将IL-6基因转染的、高分泌的IL-6的B16黑色素瘤细胞制成瘤苗。结果发现,体内注射IL-6基因转染的瘤苗后,小鼠脾脏CTL活性、NK活性及IL-2诱导的LAK活性显著升高。经IL-6基因转染瘤苗体内治疗后,荷瘤小鼠的皮下肿瘤生长显著减慢、肺转移结节数显著降低、存活期显著延长,若同时合用低剂量IL-2,则上述治疗效果更好。可见IL-6基因转染的瘤苗能有效地通过诱导机体抗肿瘤免疫功能而发挥抗肿瘤作用,与低剂量IL-2合用后,IL-6基因转染的瘤苗的抗肿瘤效果更佳。     

自体肿瘤疫苗主动特异性免疫治疗进展期肿瘤的初步研究

目的探讨自体肿瘤疫苗的作用机制及临床意义.方法30例进展期肿瘤病人术后,以自体肿瘤疫苗辅助主动免疫治疗.术后第4周开始接种(共4次,每次间隔7～10 d);接种前3天及4次接种后1 w,采集外周血,分离单个核细胞,测定CD8+-IFN-γ+,CD8+-IL-10+细胞比例及CD4+-IFN-γ+、CD4+-IL-10+细胞比例;同时采集血清检测血清IFN-γ、IL-10水平;用PPD及自体抗瘤抗原做皮肤迟发型过敏反应试验,48h后测量红斑、硬结大小(mm).临床随访.结果自体免疫苗治疗后①血清IFN-γ水平升高,高(5.98±2.40)pg/ml升至(11.20±4.76)pg/ml;而IL-10水平由(26.04±12.85)pg/ml降至(8.12±3.69)pg/ml,差异有非常显著性(P＜0.05).②CD8+-IFN-γ+双阳性细胞比例由(3.72±1.56)%升至(8.01±2.54)%;CD4+-IFN-γ+双阳性细胞比例由(4.52±1.08)%升至(7.94±2.06)%.差异有非常显著性(P＜0.05).③治疗前后病人对自体肿瘤抗原的特异性DTH反应明显增强(P＜0.01).④病人耐受性良好,无溃疡等严重副作用发生.⑤随访结果显示22例病人无瘤生存时间超3年,其中部分病人无瘤生存时间超过5年.结论①自体肿瘤疫苗可激发病人特异性细胞介导的免疫反应;②自体疫苗可改善病人的抗瘤免疫反应;③自体肿瘤疫苗主动特异性免疫治疗,对于杀灭残留癌细胞、抗转移及复发有重要作用.     

Inhibition of tumor-associated macrophages by trabectedin improves the antitumor adaptive immunity in response to anti-PD-1 therapy

A considerable proportion of cancer patients are resistant or only partially responsive to immune checkpoint blockade immunotherapy. Tumor-Associated Macrophages (TAMs) infiltrating the tumor stroma suppress the adaptive immune responses and, hence, promote tumor immune evasion. Depletion of TAMs or modulation of their protumoral functions is actively pursued, with the purpose of relieving this state of immunesuppression. We previously reported that trabectedin, a registered antitumor compound, selectively reduces monocytes and TAMs in treated tumors. However, its putative effects on the adaptive immunity are still unclear. In this study, we investigated whether treatment of tumor-bearing mice with trabectedin modulates the presence and functional activity of T-lymphocytes. In treated tumors, there was a significant upregulation of T cell-associated genes, including CD3, CD8, perforin, granzyme B, and IFN-responsive genes (MX1, CXCL10, and PD-1), indicating that T lymphocytes were activated after treatment. Notably, the mRNA levels of the Pdcd1 gene, coding for PD-1, were strongly increased. Using a fibrosarcoma model poorly responsive to PD-1-immunotherapy, treatment with trabectedin prior to anti-PD-1 resulted in improved antitumor efficacy. In conclusion, pretreatment with trabectedin enhances the therapeutic response to checkpoint inhibitor-based immunotherapy. These findings provide a good rational for the combination of trabectedin with immunotherapy regimens.     

DNA疫苗与肿瘤免疫治疗

DNA疫苗因其能诱发有效及持久的免疫反应而被广泛应用于恶性肿瘤的免疫治疗。已构建出多种肿瘤DNA疫苗,包括TAAs全长DNA疫苗、TAAs表位DNA疫苗、肿瘤融合DNA疫苗。肿瘤DNA疫苗可以激发肿瘤特异性的细胞/体液免疫反应及延长实验动物的生存期。肿瘤DNA疫苗在动物试验中的有效性为其在临床治疗中的应用提供了理论依据。     

Assessment of oxidative DNA damage in the oxyR-deficient SOS chromotest strain Escherichia coli PQ300.

The SOS chromotest is a simple short-term genotoxicity assay measuring the induction of gene sfiA in Escherichia coli K-12. The recent availability of SOS tester strains with additional mutations in DNA repair or protection systems allows testing of DNA damaging compounds for genotoxic specificity. E. coli PQ300 differs from the standard SOS tester strain PQ37 in that it contains an additional mutation in gene oxyR that renders it more sensitive to oxidative genotoxins. The generation of reactive oxygen intermediates (ROI) by hydroperoxides (H2O2, t-butyl hydroperoxide, cumene hydroperoxide), gamma-radiation, glucose oxidase, and xanthine oxidase resulted in a more vigorous SOS response in strain PQ300 compared to strain PQ37. PQ300 was also more sensitive than PQ37 for the detection of reducing agents such as ascorbic acid, cysteine, and glutathione, which also alter the redox status of the bacterial cells. However, intercalating agents (adriamycin, bleomycin, and mitomycin C) and the UV- and radiomimetic compound 4-nitroquinoline-1-oxide whose DNA damaging potential are known also to involve ROI did not show significant differences between strains PQ37 and PQ300. It is concluded that the oxyR-deficient strain PQ300 is useful for detecting certain classes of genotoxins that change the oxidative/antioxidative balance of tester bacteria in the SOS chromotest.     

CpG DNA and immunotherapy of allergic airway diseases

Atopic asthma is a highly prevalent and serious health problem for which no therapy currently offers the hope of a cure. Preindustrialized and rural populations appear relatively protected from the asthma epidemic; the hygiene hypothesis ascribes this protection to the effects of microbes and microbial products. An important immunostimulant component of microbes is DNA; bacterial DNA contains sequence motifs centred on the CpG dinucleotide, which are suppressed in mammalian DNA. Oligonucleotides containing these motifs (CpG ODN), like bacterial DNA, promote Th1 and regulatory-type immune responses. Using CpG ODN, we and others have demonstrated in murine studies that CpG ODN are effective in preventing the development of atopic airways disease. Moreover, when administered in conjunction with experimental allergen, they promote the reversal of established eosinophilic inflammation. These data suggest that CpG ODN may be a novel therapeutic tool for the treatment of atopic asthma.     

树突状细胞的抗原基因转导及其抗肿瘤免疫作用的研究

目的制备整合素靶向性腺病毒载体RGD-Adv,向树突状细胞（DCs）转导抗原基因,并研究表达特异性抗原DCs的免疫学特点和抗肿瘤效果。方法使用体外连接法构建RGD-Ad,并以流式细胞技术检测其基因转导效率,通过抗原呈递试验、CTL效应试验和小鼠体内抗肿瘤试验考察表达特异性抗原DCs诱导产生CTL效应和在体抗肿瘤作用情况。结果RGD-Adv可安全高效转导DCs,且为病毒载体剂量依赖性;表达特异性抗原DCs能有效诱导抗原特异性CTL效应,并具有显著抗肿瘤效果。结论RGD-Adv是一种理想的载体系统,通过使DCs表达特异性抗原,诱导机体特异性免疫机制,产生抗肿瘤效果,是一种极具前景的肿瘤免疫治疗方法。     

DC/C6融合瘤苗防治C6胶质瘤的实验研究

目的：探讨DC/C6融合瘤苗防治C6胶质瘤的疗效及作用机制。方法：采用PEG化学融合方法制备融合瘤苗，应用GFAP-FITC免疫荧光检查进行瘤苗的鉴定；立体定向制备大鼠颅内C6肿瘤模型，于种瘤后5 d经尾静脉注射107融合瘤细胞、107DC以及100 μL PBS，分设为A、B、C 3组，采用Log-rank对数秩检验进行生存分析，并行肿瘤标本HE染色及抗CD8Mcab免疫组化染色。结果： 融合瘤苗GFAP-FITC免疫荧光检查阳性；Log-rank生存分析对数据进行对数秩检验，结果表明A组与B、C组进行比较均有统计学意义（P<0.01）；A组晚期死亡大鼠（>31 d）HE染色见较多的炎性细胞浸润，CD8Mcab免疫组化染色阳性。结论：DC/C6融合瘤苗能够有效的发挥抗原提呈、活化T淋巴细胞的功能，CD8+T细胞参与抗胶质瘤免疫反应。     

Treatment with proteasome inhibitor bortezomib enhances antigen-specific CD8+ T-cell-mediated antitumor immunity induced by DNA vaccination

There is an urgent need to develop new innovative therapies for the control of cancer. Antigen-specific immunotherapy and the employment of proteasome inhibitors have emerged as two potentially plausible approaches for the control of cancer. In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 antigen (CRT/E7) with the proteasome inhibitor, bortezomib, for their ability to generate E7-specific immune responses and antitumor effects in vaccinated mice. We found that the combination of treatment with bortezomib and CRT/E7(detox) DNA generated more potent E7-specific CD8+ T cell immune responses and better therapeutic effects against TC-1 tumors in tumor-bearing mice compared to monotherapy. Furthermore, we found that treatment with bortezomib led to increased apoptosis of TC-1 tumor cells and could render the TC-1 tumor cells more susceptible to lysis by E7-specific CD8+ T cells. Our data have significant implications for future clinical translation.     

人免疫缺陷病毒Ⅱ型核心蛋白DNA疫苗的实验免疫研究

目的　检测HIV 2核心蛋白DNA疫苗诱导Balb c小鼠免疫应答的能力。方法　将表达HIV 2核心蛋白DNA疫苗质粒pVAXIgag肌注Balb c小鼠 ,分析CD4 、CD8 T淋巴细胞的数量、脾特异性CTL反应、血清中HIV 2的特异性抗体水平。结果　重组质粒pVAXI gag免疫组与空载体pVAXI及PBS对照组相比较差异显著 ,血清抗体滴度及淋巴细胞杀伤效应为P <0 .0 1,脾T细胞亚群的数量为P <0 .0 5。结论　HIV 2核心蛋白DNA疫苗能诱导Balb c小鼠产生特异性细胞免疫应答和体液免疫应答