Intramuscular injection of plasmid DNA encoding cottontail rabbit papillomavirus E1, E2, E6 and E7 induces T cell-mediated but not humoral immune responses in rabbits

To test the efficacy of genetic vaccination against papillomavirus infection, plasmid DNA encoding cottontail rabbit papillomavirus (CRPV) E1, E2, E6, E7 or without insert were intramuscularly injected into five groups of rabbits. Peripheral blood mononuclear cells (PBMCs) showed specific proliferation upon in vitro stimulation with E1, E2, E6 or E7 proteins in a majority of vaccinated rabbits but Western blot analysis did not detect antibodies specific for these viral proteins in rabbit serum. All rabbits grew papillomas after virus challenge and none of the rabbits showed systemic papilloma regression. These observations showed that intramuscular injection of plasmid DNA encoding CRPV E1, E2, E6 or E7 induced CD4+ T cell-mediated but not humoral immune responses, and did not result in the protection of rabbits from virus infection.     

Enhancement of human papillomavirus (HPV) type 16 E6 and E7-specific T-cell immunity in healthy volunteers through vaccination with TA-CIN,an HPV16 L2E7E6 fusion protein vaccine

TA-CIN is a vaccine that comprises the human papillomavirus (HPV) type 16 L2, E6 and E7 as a single fusion protein. In a mouse model, TA-CIN effectively prevented outgrowth of HPV16-positive tumour cells. To assess the safety and immunogenicity of TA-CIN, a dose escalating (26, 128, 533 micro g), double blind and placebo-controlled phase I study was conducted in 40 healthy volunteers. TA-CIN was administered without adjuvant by intramuscular injection on weeks 0, 4 and 8. No serious adverse events of the vaccination were reported during the study. Both IgG antibodies and proliferative responses against TA-CIN were elicited at all three doses. More importantly, T-cell immunity against the HPV16 E6 and E7 oncoproteins was detected by IFN gamma ELISPOT in 8/11 evaluable subjects vaccinated with the 533 micro g dose.     

Therapeutic efficacy of vesicular stomatitis virus-based E6 vaccination in rabbits

Millions of people worldwide are currently infected with human papillomaviruses (HPVs). A therapeutic HPV vaccine would have widespread applicability because HPV-associated lesions are difficult to treat and may progress to carcinoma. We developed three attenuated VSV recombinants expressing the cottontail rabbit papillomavirus (CRPV) early protein E6 for use as vaccines. In cultured cells, two vectors expressed different levels of the E6 protein, and one expressed a ubiquitin-E6 fusion protein. All three were tested for therapeutic efficacy in the cottontail rabbit papillomavirus (CRPV)-rabbit model. Mock vaccination had no effect on papilloma growth. In contrast, inoculation with any of the VSV-E6 vaccines reduced the rate of papilloma growth to as little as 24% the rate in the controls. In five experiments, these effects were achieved after a single immunization. Furthermore, complete papilloma regression occurred in some rabbits observed for 4 months. A VSV-based papillomavirus E6 vaccine could have significant advantages over other therapeutic HPV vaccine candidates described to date.     

Therapeutic vaccination with papillomavirus E6 and E7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model

This study was performed to test the therapeutic efficacy of overlapping long E6 and E7 peptides, containing both CD4+ T-helper and CD8+ CTL epitopes, on CRPV-induced lesions, which is an appropriate pre-clinical model for HPV diseases, including recurrent respiratory papillomatosis (RRP). Therapeutic peptide vaccination was able to significantly control wart growth (p < 0.01) and abrogate latent CRPV infection (p = 0.0006) compared to controls. Vaccination was associated with a T(H)1 T cell response, as suggested by a strong DTH skin test, antigen-specific proliferation of PBMC and a minimal IgG antibody response. Thus, this study shows promise for treatment of RRP by vaccination with long peptides.     

Identification of human papillomavirus type 53 L1, E6 and E7 variants in isolates from Brazilian women

Human papillomavirus type 53 (HPV 53), which belongs to genus Alpha, species A6, has spread among women worldwide. Although it is classified as a probably high risk type, the association between HPV 53 and the development of neoplastic cervical disease is unclear, and HPV 53 is known to be genomically diverse. We investigated 15 cases of HPV 53 genital infection in women living in the state of Rio de Janeiro that were not associated with severe intraepithelial cervical neoplasia. To trace HPV 53 variants in this geographic area, we characterized the L1, E6 and E7 genes from these isolates, and undertook a phylogenetic analysis based on multiple alignment of their L1 sequences. After amplification and sequence analysis, we identified seven different L1-E6-E7 variants and a L1 co-infected isolate, which taken together had base pair changes at 29 different positions. The co-infected sample presented overlapped peaks at two positions. We also detected two new E6 genomic variants. Several base pair changes in the E6 region resulted in amino acid changes, three of which were non-conservative. The E7 gene was the most conserved sequence among those studied; in contrast, the E6 sequence reached a maximum difference of 2.79%. None of the HPV 53 isolates corresponded to the reference type. Dichotomical branching characteristic of HPV 53 was observed in all of the trees constructed, as well as in the concatenated phylogenetic tree. Probably, these variants pointing to evolutionary process, but they not appear to keep an increasing of pathogenesis Despite the limited number of samples analyzed in our work, we noticed that the same variant was found in more than one woman. Therefore, it is possible that such variants have been circulating in the female population in the state of Rio de Janeiro for a longer time or that due to host or genetic viral factors, these variants can spread more rapidly than others.     

氢氧化铝对人乳头瘤病毒16型L2E6E7融合蛋白免疫增强作用的初步研究

目的 初步评价Al(OH)3对重组HPV16的L2E6E7融合蛋白(简称HPV16L2E6E7)的免疫佐剂作用.方法 将108只雌性C57BL/6小鼠随机分成Al(OH)3组(85.7 μg/只,肌肉注射)、HPV16L2E6E7组(120.0μg/只,肌肉注射)、A1 (OH)3+HPV16L2E6E7组[每只 Al(OH)3 85.7μg+HPV16L2E6E7120.0μg,肌肉注射],每组36只,免疫程序为0、3、7d.ELISPOT法检测IFN-γ,间接ELISA法检测IgG抗体水平.另将40只皮下接种TC-1肿瘤细胞(1×104/只)的雌性C57BL/6小鼠随机分为PBS组(100.0 μL)、Al (OH)3组(85.7μg/只,肌肉注射)、HPV16L2E6E7组(120.0μg,/只,肌肉注射)、Al(OH)3+ HPV16L2E6E7组[每只Al(OH)385.7μg+HPV16L2E6E7 120.0μg,肌肉注射],每组10只.免疫程序为0、3、7d.观察肿瘤形成时间、体积变化及成瘤率,观察期为53 d.结果 免疫后第10、14、21、28、35和42d,HPV16L2E6E7组与Al(OH)3+HPV16L2E6E7组IgG抗体滴度都高于Al(OH)3对照组,差异具有统计学意义(P均＜0.01),免疫后21、28、35、42 d Al(OH)3+ HPV16L2E6E7组特异性IgG抗体显著高于HPV16L2E6E7组(P均＜0.01);免疫后14、21、28d Al(OH)3+ HPV16L2E6E7组E7特异性IFN-γ水平显著高于HPV16L2E6E7组(P均＜0.01);小鼠抑瘤实验中,HPV16L2E6E7组抑瘤率为60％,Al(OH)3+ HPV16L2E6E7抑瘤率为40％,两组无明显差异(P＞0.05),但与对照组比较都有显著性差异(P＜ 0.01,P＜0.05).结论 初步评价Al(OH)3能与HPV16L2E6E7较好吸附,可诱导C57BL/6雌性小鼠产生更强的特异性体液和细胞免疫应答,具有免疫增强作用.     

GM-CSF enhances protective immunity to cottontail rabbit papillomavirus E8 genetic vaccination in rabbits

We have reported previously that cottontail rabbit papillomavirus (CRPV) E8 gene immunization induced strong protection against virus challenge. In this study, we primed E8 gene vaccination with mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF), a cytokine that induces differentiation and local recruitment of professional antigen-presenting cells. EIII/JC inbred rabbits were divided into four groups receiving vaccinations with the following constructs: mGM-CSF plus E8, mGM-CSF only, E8 only and vector only. After three immunizations at intervals of 3 weeks, rabbits were challenged with viral DNA at six scarified sites. Papillomas grew on all vaccinated rabbits 4 weeks after inoculation. At week 5, papillomas on four rabbits of mGM-CSF plus E8 and one of E8 only rabbits began to regress. At week 11, all the papillomas on rabbits in the GM-CSF plus E8 vaccination group regressed (regression rate = 100%); regression rates of the mGM-CSF only and E8 only vaccination groups were 50 and 25%, respectively. All papillomas on the vector immunized rabbits remained persistent until the end of the experiment (0%). Antibodies to mGM-CSF were detected in rabbit serum by Western blot. Rabbits vaccinated with E8 plus mGM-CSF or E8 only group had positive Delayed-type hypersensitivity (DTH) skin test to different E8 peptides. These results demonstrated that mGM-CSF could enhance the effects of E8 immunization in rabbits to CRPV infection through cell-mediated immune responses.     

表达人乳头瘤病毒16型E6E7重组腺病毒疫苗对小鼠的免疫和抗肿瘤效应

目的 评价HPV16 E6E7的复制缺陷型重组5型腺病毒(PK-HPV-ad5)治疗性疫苗对实验小鼠免疫应答和抗肿瘤的生物学效应.方法 使用基因重组技术构建PK-HPV-ad5疫苗,并通过小鼠免疫试验,检测小鼠总抗体和特异性IFNγ,同时将造模小鼠分成疫苗组和对照组,分别对其进行抑瘤试验、TC-1肿瘤细胞挑战试验和肿瘤切除后防复发试验.结果 HPV16 E6E7诱导的总抗体第12天的水平相对较高(1:400～1:600);3批次疫苗特异性IFNγ在第14天与对照组比较分别升高8.6、5.9和8.9倍,差异有统计学意义(t=15.721、6.967和14.342,P均＜0.01).抑瘤试验表明疫苗剂量为107IU/只时小鼠肿瘤生长率为0,与对照组比较差异有统计学意义(确切概率法,P＜0.01),3批次疫苗验证有效剂量为107IU/只时肿瘤抑制率可达80％(8/10)以上.TC-1肿瘤细胞挑战试验结果显示:小鼠先接种疫苗能引起特异性的免疫应答,并能保护90％(9/10)的小鼠免受TC-1肿瘤细胞的攻击;肿瘤切除后防止复发试验提示在注射相同剂量疫苗时,对104个/只和105个/只肿瘤细胞造模小鼠,第0、5天免疫组肿瘤复发数少于第5,8天免疫组(1/10,4/10 vs 8/10,7/10).结论 PK-HPV-ad5疫苗能诱导小鼠产生特异性的免疫应答,对抗肿瘤复发有治疗潜力.     

人乳头瘤病毒11型E7与共刺激分子CD80嵌合DNA疫苗质粒的构建及鉴定

目的:构建人乳头瘤病毒11型(HPV ll)E7与共刺激分子CDS0嵌合DNA疫苗质粒。方法:从尖锐湿疣病变组织中提取DNA制备模板,采用聚合酶链反应(PCR)技术扩增HPV ll-E7基因,经双酶切后定向克隆到pcDNA3.1(+)中,获得pcDNA3.1(+)/HPV llE7,测序鉴定后,再用PCR方法扩增去除终止密码子的E7基因片段,按上述方法插入质粒pcD-NA3.1(+)/CD80中CD80上游,构建pcDNA3.1(+)/HPV llE7-CD80融合基因真核表达质粒。结果:去除终止子的E7基因片段成功正向插入CD80上游,双向测序分析显示序列无误,pcDNA3.1(+)/HPV llE7-CD80嵌合真核表达质粒构建成功。结论:HPV ll-E7/CD80嵌合DNA疫苗质粒构建成功,为研究该疫苗在尖锐湿疣防治中的作用奠定了基础。     

Human papillomavirus type 16 E6 and E7 gene variations associated with cervical cancer in a Han Chinese population

BackgroudHuman papillomavirus type 16 (HPV16) is a high-risk HPV subtype and a potent carcinogen. The HPV16 E6 and E7 genes are considered oncogenes that play a core role in the development of cervical cancer.MethodsIn the current study, we enrolled 97 HPV16-positive cervical cancer patients (case group) and 136 HPV16-positive asymptomatic individuals (control group) in a study to analyse the association between HPV16 E6 and E7 gene variations and cervical cancer.ResultsOur results showed that three HPV16 sub-lineages (A1-A3, A4 and D3) were present; the distribution of these variants between the case and control group was not significantly different (P = 0.178). When the distribution of the HPV16 E6 and E7 gene variations was compared, the distribution of only A131C (R10R) in the E6 gene showed a different trend between the case and control groups and C749T (S63F) in the E7 gene was significantly different between the case and control groups (P = 0.071 and P = 4.861 × 10⁻¹⁰, respectively). Regarding the sub-lineages, no variations in the E6 gene were significantly different between the case and control group for the A4 (As) and A1-A3 (EUR) sub-lineages. However, the distribution of C749T (S63F) in the E7 gene was significantly different between the case and control groups for the A4 (As) and A1-A3 (EUR) sub-lineages (P = 1.815 × 10⁻⁸ and P = 0.008). In the current study, we found that the C749T (S63F) variation in the HPV16 E7 gene was associated with cervical cancer not only in the A4 (As) sub-lineage but also in the A1-A3 (EUR) sub-lineage.ConclusionOur study will provide a good reference for further functional studies of the relationship between cervical cancer carcinogenesis and the HPV16 E6 and E7 genes.     

Preclinical development of peptide vaccination combined with oncolytic MG1-E6E7 for HPV-associated cancer

Human papilloma virus (HPV)-associated cancer is a significant global health burden and despite the presence of viral transforming antigens within neoplastic cells, therapeutic vaccinations are ineffective for advanced disease. HPV positive TC1 cells are susceptible to viral oncolysis by MG1-E6E7, a custom designed oncolytic Maraba virus. Epitope mapping of mice vaccinated with MG1-E6E7 enabled the rational design of synthetic long peptide (SLP) vaccines against HPV16 and HPV18 antigens. SLPs were able to induce specific CD8+ immune responses and the magnitude of these responses significantly increased when boosted by MG1-E6E7. Logically designed vaccination induced multi-functional CD8+ T cells and provided complete sterilising immunity of mice challenged with TC1 cells. In mice bearing large HPV-positive tumours, SLP vaccination combined with MG1-E6E7 was able to clear tumours in 60% of mice and these mice were completely protected against a long term aggressive re-challenge with the TC1 tumour model. Combining conventional SLPs with the multi-functional oncolytic MG1-E6E7 represents a promising approach against advanced HPV positive neoplasia.     

Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) and cancer. A newly designed vaccine, comprising the HPV16 L2, E6 and E7 as a single fusion protein (TA-CIN), was shown to elicit HPV16-specific CTL, T-helper cells and antibodies in a pre-clinical mouse model. These immune responses effectively prevented outgrowth of HPV16-positive tumour cells in a prophylactic setting as well as in a minimal residual disease setting. CTL immunity was optimally induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine. These data provide a scientific basis for the use of TA-CIN, alone or in combination with TA-HPV in future human trials.     

HPV E6/E7 mRNA检测用于ASCUS患者诊断分流的临床研究

目的:探讨人乳头瘤病毒(HPV)E6/E7mRNA检测用于未明确诊断意义的不典型鳞状上皮细胞(ASCUS)诊断结果的判定价值。方法:对160例薄层液基细胞学检查诊断为ASCUS者进行HPV E6/E7mRNA和高危型HPV(HR-HPV)DNA检测,结合病理学诊断资料进行统计学分析。结果:160例中病理结果为宫颈上皮内瘤变Ⅱ级(CINⅡ)及以上级别者的HPV E6/E7mRNA阳性率明显高于CINⅡ以下级别者,差异有统计学意义(P0.05),但与HR-HPV DNA检测结果无统计学差异。HPV E6/E7 mRNA检测CINII及以上级别的灵敏度(69.2%)与HR-HPV DNA检测相比无统计学差异(P0.05);特异度(73.9%)高于HR-HPV DNA检测(61.9%),差异有统计学意义(P0.05)。结论:HPV E6/E7 mRNA检测可作为ASCUS者是否需要进行阴道镜检查的一项指标依据。     

人乳头瘤病毒 E6／E7 mRNA检测在宫颈癌筛查中的意义

目的：评估人乳头瘤病毒（HPV）E6／E7mRNA检测在宫颈癌筛查中的意义。方法对3381名行机会性宫颈癌筛查的女性分别行液基细胞学（TCT）、HPV DNA及HPV E6／E7mRNA检查,其中371例结果可疑者行阴道镜下活检。分别记录其TCT、HPV DNA及HPV E6／E7mRNA检测结果及活检病理结果等,采用SPSS 19．0软件进行数据分析。结果人群筛查中HPV E6／E7mRNA的总阳性率为16．47％（557／3381）;炎症患者与宫颈上皮内瘤变（CIN）Ⅰ级患者HPV E6／E7mRNA的阳性率分别为60．73％（116／191）和68．60％（83／121）,差异无统计学意义（F＝1．98,P＞0．05）;炎症患者与CIN Ⅰ级患者mRNA表达量分别为7781．26±2686．13和7498．47±2860．08 copy／mL,亦无显著差异（ t＝0．07,P＞0．05）,而CIN Ⅱ-Ⅲ级患者HPV E6／E7mRNA的阳性率为89．10％（49／55）,mRNA表达量为30716．66±10281．89,均显著高于炎症患者（χ2＝15．55,t＝2．19）及CINⅠ患者（χ2＝8．47,t＝2．18）,差异均有统计学意义（P＜0．05）;不同活检病理分级（≤CIN Ⅰ或≥CIN Ⅱ）中,只有HPV E6／E7mRNA的检测结果有显著性差异（χ2＝15．45,P＜0．05）;对于宫颈CIN Ⅱ以上病变的诊断,HPV E6／E7mRNA的敏感度最高（89．83％）;TCT特异度最高（62．18％）。 HPV E6／E7mRNA最佳工作点为1637．48copy／mL,其诊断宫颈高级别上皮内瘤变的敏感度为79．70％,特异度为63．50％。 ROC曲线下面积为0．75（P＜0．05）。结论 HPV E6／E7mRNA检测诊断宫颈高级别上皮内瘤变具有较高的敏感性和特异性,在宫颈癌筛查具有较为重要的意义。     

人乳头瘤病毒E6/E7 mRNA检测在宫颈上皮内瘤变1预后中的预测价值

目的探讨人乳头瘤病毒(HPV) E6/E7 mRNA检测在宫颈上皮内瘤变(CIN)1患者预后中的预测价值。 方法选取2013年7月至2014年10月,于烟台市烟台山医院妇科经阴道镜下宫颈活组织病理学检查,首次确诊为CIN1的107例患者为研究对象。所有受试者均于首次确诊为CIN1前1个月内,同时接受宫颈液基薄层细胞学检查(TCT)、HPV DNA基因分型及HPV E6/E7 mRNA检测;于阴道镜下宫颈活组织病理学检查确诊为CIN1后,对其进行为期2年的随访,每6个月随访1次。随访检查项目包括宫颈TCT、HPV DNA基因分型及HPV E6/E7 mRNA检测,并对可疑病变者予以阴道镜下宫颈活组织病理学检查。根据随访结束时受试者的TCT、HPV DNA基因分型、HPV E6/E7 mRNA检测结果及阴道镜下宫颈活组织病理学检查结果,将本研究受试者分为转阴组与持续或进展组。采用χ²检验,对2组受试者的HPV E6/E7 mRNA阳性率进行比较;采用Mann-Whitney U秩和检验,对2组HPV E6/E7 mRNA表达水平进行比较;计算3种宫颈病变筛查方法对于预测CIN1预后的敏感度和特异度;绘制HPV E6/E7 mRNA表达水平预测CIN1预后的受试者工作特征(ROC)曲线,计算ROC曲线下面积(ROA-AUC),根据约登指数最大原则,确定HPV E6/E7 mRNA表达水平预测CIN1预后的最佳临界值,同时计算其敏感度和特异度。本研究遵循的程序符合烟台市烟台山医院伦理委员会所制定的标准,获得该伦理委员会批准,并与受试者均签署临床研究知情同意书。 结果①根据随访结束时受试者的TCT、HPV DNA基因分型、HPV E6/E7 mRNA检测结果及阴道镜下宫颈活组织病理学检查结果,将本研究受试者分为转阴组(n＝63,随访结束时,TCT、HPV DNA基因分型及HPV E6/E7 mRNA检测结果均为正常,或阴道镜下宫颈活组织病理学检查结果为阴性),持续或进展组(n＝44,随访结束时,阴道镜下宫颈活组织病理学检查结果为CIN1及以上)。2组患者的年龄等一般临床资料比较,差异均无统计学意义(P>0.05)。②持续或进展组受试者确诊为CIN1时的HPV E6/E7 mRNA阳性率及其中位表达水平分别为61.9%(39/63)和3 738.4 copy/mL,转阴组分别为81.8%(36/44)和583.5 copy/mL,持续或进展组HPV E6/E7 mRNA阳性率及其中位表达水平均显著高于转阴组,并且差异均有统计学意义(χ²＝4.901,P＝0.027;U＝821.000,P<0.001)。③HPV-16/18阳性患者CIN持续或进展率为50.8%(31/61),显著高于HPV-16/18阴性者的28.3%(13/46),并且差异有统计学意义(χ²＝5.512,P＝0.019)。HPV E6/E7 mRNA阳性患者CIN持续或进展率为48.0%(36/75),显著高于HPV E6/E7 mRNA阴性者的25.0%(8/32),并且差异亦有统计学意义(χ²＝4.901,P＝0.027)。TCT、HPV DNA基因分型及HPV E6/E7 mRNA定性检测对于预测CIN1患者预后的敏感度分别为50.0%、70.5%、81.8%,特异度分别为66.7%、52.4%、38.1%。④HPV E6/E7 mRNA表达水平预测CIN1患者预后的ROC曲线分析结果显示,HPV E6/E7 mRNA表达水平预测CIN1持续或进展的ROC-AUC为0.704(95%CI：0.601～0.806,P<0.001),HPV E6/E7 mRNA表达水平预测CIN持续或进展的最佳临界值为2 724.0 copy/mL,此时其预测CIN1持续或进展的敏感度为54.5%,特异度为81.0%。 结论HPV E6/E7 mRNA定性检测结果对于预测CIN1患者预后的敏感度较高,而其定量检测结果对于预测CIN1患者预后的特异度较高。HPV E6/E7 mRNA检测可能对CIN1患者预后的预测具有一定临床价值。     

高危型HPV E6/E7 mRNA与宫颈癌相关性分析

目的 探讨高危型HPV E6/E7 mRNA检出率与宫颈癌的相关性,为临床防治宫颈癌提供依据。方法 选择2015年收治的100例宫颈癌患者为A组,同期100例健康体检者为B组,采用荧光定量PCR检测入组患者高危型HPV E6/E7 mRNA和病理学检查,比较两组患者HPV E6/E7感染率和荧光定量PCR检查效率,分析HPV E6/E7感染与宫颈鳞状上皮病变的相关性。结果 A组阳性76例,阳性率为76.0%;B组阳性13例,阳性率为13.0%;A组阳性率高于B组,差异有统计学意义（χ²=24.522,P<0.001）。两组阳性预测值和阴性预测值比较,差异无统计学意义（P>0.05）。宫颈癌患者HPV E6/E7 mRNA阳性率（76.0%）高于高度宫颈鳞状上皮病变者（26.1%）、低度宫颈鳞状上皮病变者（17.6%）和非典型鳞状上皮细胞者（6.7%）,差异有统计学意义（χ²=7.615,P=0.001; χ²=9.114,P=0.001; χ²=18.241,P<0.001）。结论 宫颈癌患者HPV E6/E7mRNA检出率高,且随宫颈鳞状上皮病变加重,其HPV E6/E7 mRNA阳性率越高。     

人乳头瘤病毒16E7 DNA疫苗微针给药效果研究

目的 探讨DNA疫苗微针给药方法的有效性.方法 构建pcDNA3.1-HPV16E7重组质粒,在体外透皮条件下,观察pcDNA-HPV16E7皮肤透过量,再利用微针给药方式免疫BALB/c小鼠,分三组(实验组、空载体对照组、阴性对照组),每组10只,每2周免疫1次,共3次,每次免疫剂量为200 μg/只,对照组参照实验组进行,最后1次免疫后2周采血,分别分离血清和淋巴细胞,用间接免疫荧光试验检测小鼠的体液免疫功能,用淋巴细胞转化试验检测小鼠的细胞免疫功能.结果 体外透皮试验显示DNA疫苗微针给药可以透过皮肤,而且透过量随着时间的延长而逐步增加,在第30小时时可以达到0.738 19 mg/cm2;通过微针给药方法DNA疫苗可以诱导小鼠产生特异性抗体,淋巴细胞转化试验显示:实验组(平均淋巴细胞转化率为47.25%)和阴性对照组(平均淋巴细胞转化率为30.00%)之间比较,χ2=12.903,P＜0.001,差异有显著统计学意义;空载体组(平均淋巴细胞转化率为43.00%)和阴性对照组之间比较,χ2=7.292,P=0.007,差异有显著统计学意义;实验组和空载体组之间比较,χ2=0.817,P=0.366,差异无统计学意义.结论 HPV16E7 DNA疫苗经微针给药可以透过皮肤吸收并诱导小鼠产生体液免疫和细胞免疫反应.     

Variations of human papillomavirus type 58 E6, E7, L1 genes and long control region in strains from women with cervical lesions in Liaoning province, China

Infection with certain types of human papillomaviruses (HPVs) is a risk factor for the development of cervical cancer. HPV type 58 (HPV 58) is prevalent among Chinese women. The intratype sequence variants differ in oncogenic potential and their prevalences vary across geographic regions. The objective of this study was to analyze the variations of HPV 58 E6, E7, L1 genes and long control region (LCR) in a large samples collected from northeastern Chinese women with cervical lesions. A total of 2938 cervical samples were collected and tested for HPV type using a chip hybridization assay. The E6, E7, L1 genes and LCR of HPV 58 strains were amplified and the amplicons were subjected to direct nucleotide sequencing for variation identification. A total of 235 specimens were HPV 58 positive. High proportions of HPV 58 E6 (83.8%), E7 (76.7%), L1 (90.8%) genes and LCR (91.4%) variants were identified in strains from Chinese women. The most frequently observed variations were C307T (52.4%) in E6, T744G (74.9%) in E7, A6014C (56.9%) in L1 genes and C7266T, A7714G (55.2%) in LCR. For the E6 gene, nine nucleotide variations were identified. Among them, the A140G (T11A), A184C (E25D), G266C (V53L) and A313G were novel variations. Sequencing of the E7 gene revealed four typical nucleotide changes: G761A (G63D), G694A (G41R), T803C (V77A) and T744G. In the L1 gene, 39 nucleotide variations and 13 amino acid substitutions were identified. Among these mutations, 21 variations are reported here for the first time. Lineage A of HPV 58 was found in 142 of 174 strains (81.6%). The most prevalent HPV 58 variants in Chinese northeastern women belongs to lineage A. Novel variations in E6 and L1 genes were also reported. These findings provide new data regarding E6 and L1 gene variations of HPV 58 from women in northeast China.     

A DNA vaccine based on a shuffled E7 oncogene of the human papillomavirus type 16 (HPV 16) induces E7-specific cytotoxic T cells but lacks transforming activity.

Vaccination with oncogene-derived DNA for anti-cancer treatment carries a risk of de-novo tumor induction triggered by the persisting recombinant DNA. We hypothesized that an oncoprotein whose primary sequence has been rearranged ('shuffled') to maintain all possible T cell epitopes still induces cytotoxic T cells against the authentic protein but is devoid of transforming properties. As a model antigen, we used the E7 oncoprotein of the human papillomavirus (HPV) type 16, the major cause of cervical cancer. We have generated an artificial E7 molecule in which four domains were rearranged and, in order to maintain all possible T cell epitopes, certain sequences were duplicated. Upon transfection of this shuffled E7 gene (E7SH) into RMA cells, presentation of an E7 Db-restricted T cell epitope was shown by an E7-specific CTL line in vitro. Immunization of C57BL/6 mice with E7SH DNA induced E7-specific CTL and also conveyed protection against E7-positive syngeneic tumor cells. No transforming activity of E7SH DNA in NIH3T3 cells was detected, as determined by focus formation, induction of S-phase under conditions of serum deprivation and degradation of endogenous pRB. Our results suggest that DNA shuffling may become a promising concept for DNA-based anti-cancer vaccines.