HPV16E7-HSP90 DNA疫苗治疗宫颈癌及其免疫机制的研究

摘 要：［目的］ 探讨HPV16E7-HSP90 DNA疫苗用于治疗宫颈癌的疗效及其可能的免疫机制。［方法］ 建立TC-1宫颈癌细胞小鼠模型,负瘤第5d开始,随机将小鼠分为HPV16E7-HSP90 DNA疫苗组与对照组：DNA疫苗组给予DNA溶液100μl（5μg）,对照组给予磷酸盐缓冲溶液(PBS)100μl,两组均肌肉注射,每3d 1次,共4次。自负瘤第5d开始至治疗完成第8d,每3d测量肿瘤体积,绘制肿瘤生长曲线。治疗结束第8d,免疫组化方法检测小鼠肿瘤组织中CD8+ T 细胞、肿瘤细胞凋亡分子FAS的表达情况,TUNEL法检测负瘤小鼠肿瘤细胞的凋亡率。［结果］ HPV16E7-HSP90 DNA疫苗组的肿瘤生长速度明显低于PBS对照组(P＜0.05)。HPV16E7-HSP90 DNA疫苗组与对照组的小鼠宫颈癌组织中CD8+ T细胞表达的光密度平均值（MOD）分别为（8.76±1.27）vs (5.70±1.40)(P＜0.05);肿瘤细胞FAS表达的MOD值分别为(8.73±0.99) vs (5.74±1.47)(P＜0.05) ;小鼠肿瘤组织中CD8+ T 细胞与FAS的表达呈正相关(P＜0.01);HPV16E7-HSP90 DNA疫苗组与对照组小鼠肿瘤细胞的凋亡率分别为57.59% vs 13.11%(P＜0.05)。［结论］ HPV16E7-HSP90 DNA疫苗可上调肿瘤细胞FAS的表达,促进肿瘤细胞凋亡,激活机体的CD8+ T细胞的免疫反应,从而产生明显的抗肿瘤效应,有望于未来用于宫颈癌及癌前病变的免疫治疗。     

HPV16E6、E7治疗性DNA疫苗强化及安全策略

HPV16治疗性DNA疫苗因具有制备简单、稳定、持续时间长、纯度高、可大规模制备等优点而受到人们的关注，但其安全性和免疫效率低是其致命弱点。许多方案已用于提高疫苗的安全性和免疫原性，如与结核分枝杆茵热休克蛋白70(HSP70)基因融合、与蛋白转运载体基因融合、突变某些功能基因、构建“自杀性”DNA疫苗等。现就其安全和强化策略进行综述，为研制安全高效DNA疫苗奠定基础。     

HPV16E6、E7治疗性DNA疫苗强化及安全策略

HPV16治疗性DNA疫苗因具有制备简单、稳定、持续时间长、纯度高、可大规模制备等优点而受到人们的关注,但其安全性和免疫效率低是其致命弱点.许多方案已用于提高疫苗的安全性和免疫原性,如与结核分枝杆菌热休克蛋白70(HSP70)基因融合、与蛋白转运载体基因融合、突变某些功能基因、构建"自杀性"DNA疫苗等.现就其安全和强化策略进行综述,为研制安全高效DNA疫苗奠定基础.     

HPV16Z-Hsp65-E6／E7无佐剂重组蛋白疫苗的研究

目的：研究治疗性HPV16Z-Hsp65-E6／E7无佐剂重组蛋白疫苗的抗肿瘤活性。方法：通过淋巴细胞增殖实验和细胞毒性杀伤实验研究该疫苗激发的细胞免疫反应及反应强度；观察该疫苗对小鼠TC-1肿瘤细胞移植瘤的体内治疗作用和对小鼠生存期的影响。结果：重组蛋白疫苗免疫小鼠后，小鼠脾淋巴细胞与该疫苗体外混合培养增殖明显，并可特异性地在体外杀伤TC-1细胞；体内抑瘤试验显示该疫苗对HPV16病毒转化的TC-1细胞小鼠移植瘤的生长有显著的抑制作用。结论：该疫苗能激发特异性细胞免疫反应；显著抑制HPV16转化的TC-1肿瘤细胞生长。     

HPV16E7-HSP70 融合蛋白在大肠杆菌中表达及纯化

[目的]在大肠杆菌中表达HPV16E7-HSP70融合蛋白并进行纯化和复性,为进一步研究HPV16E7-HSP70融合蛋白抗喉癌免疫活性奠定基础.[方法]用构建的原核表达质粒pET28a HPV16E7-HSP70转化大肠杆菌BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达;通过超声波裂解细菌,高浓度尿素裂解包涵体和Ni2+离子金属螯合亲和层析纯化目的蛋白;并对融合蛋白进行复性;用SDS-PAGE及Western Blot进行分析鉴定.[结果]SDS-PAGE 分析显示有分子量约为90kD的融合蛋白表达,表达产物以包涵体形式存在,表达量可达30％;纯化后目的蛋白的纯度达到95％;经Western Blot鉴定复性后的融合蛋白能与抗HPV 16E7抗体、抗HSP70抗体特异性结合.[结论]HPV16E7-HSP70融合蛋白能够在体外大肠杆菌中表达获得.     

地方株HPV16E7基因疫苗诱发的小鼠免疫反应

目的评价地方株HPV16E7基因疫苗诱导的免疫反应。方法从先期构建的地方株HPV16E7基因重组质粒pGEM-T-E7经限制性核酸内切酶酶切,获得E7基因亚克隆到真核表达载体pcDNA3.1(-)中,构建地方株pcDNA-E7基因疫苗,转染CHO细胞,通过IFA试验验证E7蛋白的表达。用pcDNA-E7肌注方法免疫6周龄BALB/c小鼠,同时设pcDNA空载体为阴性对照,于第1天、第15天和第43天共免疫3次,用IFA和MTT法分析基因免疫在小鼠体内诱导的免疫应答。结果pcDNA-E7免疫小鼠后能检测出特异的抗体,而原载体免疫后不能产生特异抗体;但2组刺激淋巴细胞增殖的能力差异不显著。结论地方株HPV16E7基因疫苗可诱导产生小鼠免疫反应。     

HPV16型重组疫苗的构建及其抗肿瘤效果

背景与目的大量研究表明,宫颈癌的发生与高危型人乳头状瘤病毒(humanpapillomavirus,HPV)的感染密切相关,其中以HPV16为主。本研究以非复制型重组痘苗病毒为载体,构建共表达HPV16L1、L2、E67蛋白的重组非复制型痘苗病毒,并检测其免疫效果。方法以痘苗病毒为载体,利用同源重组技术构建共表达HPV16L1、L2、E67基因的重组痘苗病毒NTVJE67CKL1L2。用该病毒免疫C57BL/6小鼠后,检测其特异性抗体和特异性的细胞毒性T淋巴细胞(cytotoxicTlymphocyte,CTL);以TC-1肿瘤细胞攻击经免疫后的小鼠,观察其免疫保护效果。结果经斑点杂交结果显示重组病毒基因组中有L1、L2、E6、E7基因插入;经Westernblot检测,重组病毒能稳定表达HPV16L1、L2、E67蛋白。动物实验结果表明,NTVJE67CKL1L2在C57BL/6小鼠体内可诱发L1、L2、E67特异性抗体产生;可诱发产生针对TC-1细胞的特异性的CTL反应;加强免疫后的小鼠能耐受1×104个TC-1肿瘤细胞的攻击。结论非复制型重组痘苗病毒NTVJE67CKL1L2可以作为对HPV16相关肿瘤及其癌前病变进行免疫治疗的候选疫苗。     

基因枪介导的neu基因DNA疫苗抗肿瘤作用的实验研究

目的 探讨DNA疫苗pWRG-neu的皮内免疫,对高表达neu基因的小鼠移植瘤生长和转移的抑制作用。方法 向小鼠黑色素瘤B16F10细胞系转染pcDNA-neu,用有限稀释法筛选一株高表达neu基因的细胞株B16F10-neu。在基因枪介导下,向C57BL／6小鼠导入DNA疫苗pWRG-neu,通过观察免疫动物的生存期,评价DNA疫苗的抗肿瘤作用。分离免疫动物脾细胞,经自体淋巴细胞混合培养实验,分析DNA疫苗体内免疫后机体的CTL应答。结果 筛选到一株高表达neu基因的B16F10-neu细胞株,转基因过程和外源基因的表达没有改变细胞系的增殖特性。用基因枪轰击,进行DNA疫苗pWRG-neu皮内免疫,对小鼠黑色素瘤B16F10-neu进行预防、治疗和抗转移的实验研究,结果表明,DNA疫苗的免疫能够明显推迟移植瘤的生长,延长小鼠生存期,获得明显的抗肿瘤效果。DNA疫苗免疫后可诱导小鼠脾淋巴细胞CTL活性。结论 基因枪介导的DNA疫苗pWRG-neu经皮内免疫,能够有效的诱导机体的细胞免疫应答,预防和治疗小鼠移植瘤的发生,并有一定的预防肿瘤肺转移的作用。     

HPV-16E6/E7在宫颈癌发生机制及疫苗中的研究进展

宫颈癌(cervicalcancer, CC)的病死率位于全球妇女癌症死亡率的第2位, 而人乳头瘤病毒(humanpapil lomaviruses, HPV)感染是宫颈癌发生的首要因素.     

宫颈癌组织中HPV_(16)DNA基因同源序列的检测

206例不同病理诊断的宫颈脱落细胞和宫颈组织DNA,同探针杂交,检测HPV_(16)DNA同源性序列。结果表明:HPV_(16)DNA探针同宫颈癌脱落细胞杂交(65.2％)和同宫颈癌组织DNA杂交(66.7％),其杂交率无显著差别(P>0.05)。宫颈炎脱落细胞DNA和宫颈炎组织DNA与HPV_(16)DNA探针杂交率间的差异也无统计学意义。然而,宫颈癌和宫颈炎之间无论是脱落细胞或是组织DNA同探针杂交有显著差异(前者为65.2—66.7％,后者为26.1—28.5％)P<0.01。宫颈癌和宫颈炎DNA经酶切电泳后,Southern blot杂交结果提示HPV_(16)DNA整合在癌组织基因组中。     

宫颈癌及其癌前病变组织中HPV16 E6、HPV16 E7蛋白的表达及其意义

目的 探讨宫颈癌及其癌前病变组织中HPV16E6、E7蛋白的表达及其意义。方法 采用免疫组化SP法对15例正常宫颈、25例宫颈上皮内瘤变（C1N）以及61例浸润性宫颈癌组织进行了HPVl6E6、HPVl6E7蛋白表达的检测。结果在正常宫颈、CINI～Ⅱ、CINⅢ及浸润性宫颈癌中，HPVl6E6蛋白的阳性表达率分别为0（0／15）、7．14％（1／14）、36．36％（4／11）、59．02％（36／61）；CINⅢ和浸润性宫颈癌中HPVl6E6蛋白阳性表达率明显高于正常宫颈组织和CINI～Ⅱ（P〈0．05）；在高、中、低分化宫颈癌中，HPVl6E6蛋白阳性表达率分别为45．45％（5／11）、77．78％（14／18）、53．13％（17／32）；HPVl6E6蛋白在不同分化程度的宫颈癌组织中的阳性表达率有显著性差异（P〈0．05），但HPVl6E6蛋白表达与官颈癌组织分化程度无明显相关性（ys=0．123），HPVl6E6蛋白阳性表达率与宫颈癌临床分期和淋巴结转移无关（P〉0．05）。HPVl6E7蛋白在正常宫颈上皮、CINI-Ⅱ、CIN Ⅲ及浸润性富颈癌组织中的阳性表达率分别为20．00％（3／15）、42．86％（6／14）、63．64％（7／11）、57．38％（35／61），HPVl6E7蛋白在不同宫颈组织中的阳性表达率无明显差异（P=0．05）；HPVl6E7蛋白的表达与富颈癌临床分期、淋巴结转移和组织分化均无关（P〉0．05）。结论 HPVl6E6蛋白的检测有可能作为宫颈癌前病变转归的指标。     

Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7

To allow vaccination irrespective of HLA type, DNA vaccines encoding full-length antigens are required. However, here, we demonstrate that the immunogenicity of DNA vaccines encoding the full-length human papillomavirus (HPV) type 16 E7 and E6 proteins is highly reduced compared to vaccines encoding only the immunodominant epitope. Furthermore, the low remaining immunogenicity is essentially lost for both E7 and E6 when a nononcogenic "gene-shuffled" variant is utilized. To address these issues, we tested whether alterations in transgene design can restore the immunogenicity of full-length and gene-shuffled DNA vaccines. Remarkably, genetic fusion of E7 with tetanus toxin fragment C (TTFC) resulted in a dramatic increase in immunogenicity both for the full-length and the gene-shuffled version of E7. Moreover, the TTFC fusion vaccines were more immunogenic than a vaccine encoding a fusion of E7 and mycobacterial heat shock protein-70, which has recently been tested in a clinical trial. Interestingly, vaccination with these TTFC fusion vaccines also resulted in extremely persistent T-cell responses. The E7-specific CD8(+) T cells induced by TTFC fusion vaccines were functional in terms of IFN-γ production, formation of immunological memory, in vivo cytolytic activity and tumor eradication. Finally, we show that genetic fusion with TTFC also improves the immunogenicity of a gene-shuffled E6 DNA vaccine. These data demonstrate that genetic fusion with tetanus toxin fragment C can dramatically improve the immunogenicity of full-length and gene-shuffled DNA vaccines. The DNA fusion vaccines developed here will be evaluated for the treatment of HPV-positive carcinomas in future studies.     

三棱、莪术提取物修饰的肿瘤细胞疫苗的非特异性抗瘤实验研究

目的:研究SE(三棱、莪术)提取物修饰的肿瘤细胞疫苗对B16小鼠恶性黑色素瘤的抗瘤效应。方法:用丝裂霉素等对K562肿瘤细胞进行灭活处理,用SE提取物进行修饰异构,经SE提取物修饰的肿瘤细胞(K562)作为瘤苗免疫小鼠,14天后再接种B16细胞,观察其抗瘤效应。取小鼠脾细胞制备LAK细胞,将LAK细胞注入小鼠腹腔,观察小鼠的存活时间。结果:SE修饰的瘤苗组存活时间为(52.71±8.26)天,与单纯瘤苗组、单纯SE中药组相比,瘤苗免疫后的小鼠存活期明显延长,有显著性差异(P<0.05);SE中药对照组治疗后其LAK细胞及NKC对B16、S180的杀伤效应明显低于SE修饰的瘤苗组(P<0.05),而正常对照组的LAK细胞对B16、S180的杀伤效应低于SE中药对照组(P<0.05);利用SE中药修饰的瘤苗免疫长期存活的小鼠脾细胞制备LAK细胞,较同龄未免疫小鼠的脾细胞制备的LAK细胞具有更强的抗瘤性。生存时间实验组(SE中药+K562+LAK)为57.26天,均长于其它对照组(P<0.05)。结论:SE中药修饰肿瘤细胞疫苗可以明显增强对B16的抗瘤效应。     

宫颈癌细胞系中HPV16 E6、E7及E6/E7基因对STK31基因甲基化状态及表达的影响

背景与目的：丝氨酸/苏氨酸蛋白激酶31(serine/threonine kinases 31,STK31)基因在人类多种癌症中扮演重要角色,且STK31基因的表达受其启动子及第一外显子区甲基化状态的影响;病毒感染与肿瘤组织中某些抑癌基因启动子区高甲基化有关。本研究旨在探讨宫颈癌细胞系中HPV16 E6、E7及E6/E7癌基因对STK31基因甲基化状态及表达的影响,以及不同种类甲基转移酶(DNA methyltransferases,DNMTs)基因在STK31基因甲基化中的潜在作用。方法：构建外源性HPV16 E6、E7以及E6/E7基因共表达慢病毒,分别感染人乳头瘤病毒(human papillomavirus,HPV)阴性宫颈癌细胞系HT-3及C33A,获得稳定转染的细胞系;采用亚硫酸氢盐基因组测序法(bisulfite genomic sequencing,BGS)和甲基化特异性PCR (methylation-specific PCR,MSP)检测3种HPV阳性宫颈癌细胞系HeLa、SiHa和CasKi以及HPV阴性宫颈癌细胞系HT-3和C33A转染前后STK31基因的甲基化状态;RT-PCR及蛋白［质］印迹法(Western blot)检测上述宫颈癌细胞系中STK31基因的表达以及DNMT1、DNMT2、DNMT3a、DNMT3b和DNMT3L基因在HPV16转染前后宫颈癌细胞系及HPV阳性、HPV阴性宫颈癌组织中的表达情况。结果：外源性HPV16 E6、E7以及E6/E7基因可在HPV阴性宫颈癌细胞系中稳定表达。3种HPV阳性细胞系HeLa、SiHa和CasKi中STK31基因呈低甲基化状态,STK31 mRNA及蛋白质表达阳性;2种HPV阴性细胞系HT-3、C33A中STK31基因则表现为高甲基化状态,STK31 mRNA及蛋白质表达缺失;与未感染慢病毒HT-3和C33A细胞系比较,外源性HPV16 E7以及E6/E7表达的HT-3和C33A细胞系STK31基因甲基化程度降低,其mRNA及蛋白质重新表达。DNMT1、DNMT3a和DNMT3b基因在HT-3E6/E7和C33AE6/E7细胞系中mRNA水平分别高于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(P<0.001)。DNMT1、DNMT3a和DNMT3b基因的mRNA水平在HPV16阳性宫颈癌组织中的表达高于其在HPV阴性宫颈癌组织中的表达,差异有统计学意义(t=5.997,P<0.001;t=6.743,P<0.001;t=7.926,P<0.001)。DNMT2在HT-3E6/E7和C33AE6/E7细胞系中mRNA表达水平分别低于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(t=7.451,P<0.001;t=2.451,P<0.05);DNMT2基因转录水平在HPV16阳性宫颈癌组织中低于HPV阴性宫颈癌组织(t=9.134,P<0.001)。DNMT3LmRNA表达水平在宫颈癌细胞系转染前后及HPV阴阳性宫颈癌组织中的差异无统计学意义(P>0.05)。结论：HPV感染可导致STK31基因启动子及第1外显子区甲基化状态降低,低甲基化状态促进该基因表达。STK31基因的表达受其启动子及第1外显子区甲基化状态的调控。HPV16 E7、E6/E7基因可能通过影响DNMT2的表达参与调控癌基因STK31基因启动子及第1外显子区甲基化状态。     

Human papillomavirus type 16 E7 oncoprotein causes a delay in repair of DNA damage

Background and purposePatients with human papillomavirus related (HPV+) head and neck cancers (HNCs) demonstrate improved clinical outcomes compared to traditional HPV negative (HPV−) HNC patients. We have recently shown that HPV+ HNC cells are more sensitive to radiation than HPV− HNC cells. However, roles of HPV oncogenes in regulating the response of DNA damage repair remain unknown.Material and methodsUsing immortalized normal oral epithelial cell lines, HPV+ HNC derived cell lines, and HPV16 E7-transgenic mice we assessed the repair of DNA damage using γ-H2AX foci, single and split dose clonogenic survival assays, and immunoblot. The ability of E7 to modulate expression of proteins associated with DNA repair pathways was assessed by immunoblot.ResultsHPV16 E7 increased retention of γ-H2AX nuclear foci and significantly decreased sublethal DNA damage repair. While phospho-ATM, phospho-ATR, Ku70, and Ku80 expressions were not altered by E7, Rad51 was induced by E7. Correspondingly, HPV+ HNC cell lines showed retention of Rad51 after γ-radiation.ConclusionsOur findings provide further understanding as to how HPV16 E7 manipulates cellular DNA damage responses that may underlie its oncogenic potential and influence the altered sensitivity to radiation seen in HPV+ HNC as compared to HPV− HNC.     

The combination of TLR‐9 adjuvantation and electroporation‐mediated delivery enhances in vivo antitumor responses after vaccination with HPV‐16 E7 encoding DNA

Therapeutic DNA vaccination is an attractive adjuvant option to conventional methods in the fight against cancer, like surgery radiotherapy and chemotherapy. Despite strong antitumor effects that were observed in small animals with different antigens, DNA‐based vaccines remain weakly immunogenic in large animals and primates compared to protein‐based vaccines. Here, we sought to enhance the immunogenicity of a therapeutic nontransforming cervical cancer DNA vaccine (HPV‐16 E7SH) by introduction of a highly optimized CpG cassette into the plasmid backbone as well as by an optimized DNA delivery using an advanced electroporation (EP) technology. By integrating the means for agent administration and EP into a single device, this technology enables a simple, one‐step procedure that facilitates reproducibility. We found that highly optimized CpG motifs alone triggers an enhanced IFN‐γ and granzyme B response in Elispot assays as well as stronger tumor regression. Furthermore, these effects could be dramatically enhanced when the CpG cassette containing plasmid was administered via the newly developed EP technology. These data suggest that an optimized application of CpG‐enriched DNA vaccines may be an attractive strategy for the treatment of cancer. Collectively, these results provide a basis for the transfer of preclinical therapeutic DNA‐based immunization studies into successful clinical cancer trials.     

Naturally processed and HLA-B8-presented HPV16 E7 epitope recognized by T cells from patients with cervical cancer

Several major histocompatibility complex (MHC) alleles have been reported to present peptides derived from the HPV16 E7 oncoprotein to T cells. We describe an overrepresentation of the HLA-B8 allele (28.44%) in cervical cancer patients as compared to the MHC class I allele frequency in a local healthy control population (18.80%) and the identification of an HLA-B8-binding peptide TLHEYMLDL (HPV16 E7(7-15)), which is able to drive HPV16 E7-specific and MHC class I-restricted T-cell responses in peripheral blood lymphocytes from healthy individuals. TLHEYMLDL-specific T cells recognize the naturally processed and presented peptide on HPV16+ cervical cancer cells transfected with the HLA-B8 gene defined by IFN-gamma production. This peptide epitope is also recognized by freshly harvested tumor-infiltrating T cells or T cells from tumor-draining lymph nodes from patients with cervical cancer determined by flow cytometry as well as by tetramer in situ staining. HLA-B8-restricted HPV E7(7-15)-specific T cells reside predominantly in the CD8+ CD45RA+ CCR7+ precursor or in the differentiated CD8+ CD45RA+ CCR7- T-cell population.