HPV16 E6/E7与结核杆菌HSP70 N端重组DNA免疫对BALB/C小鼠免疫应答的影响

目的 :探讨HPV16 (人乳头状瘤病毒 16 )E6 /E7(原癌基因E6 /E7型 )以及与HSP70N端重组DNA免疫小鼠后 ,小鼠体内免疫应答的变化。方法 :以HPV16E6 /E7为基础的DNA疫苗免疫小鼠 ,并经酶联免疫吸附实验 (ELISA)及逆转录聚合酶链反应 (RT PCR)技术检测小鼠脾淋巴细胞产生的TH1/TH型细胞因子及血清抗体。结果 :HPV16DNA疫苗免疫组小鼠 ,其脾淋巴细胞IL 2及IFNγ的分泌量明显较对照组增加 (P <0 0 1) ;HPV16E6 /E7与HSP70N端重组后疫苗免疫小鼠 ,E6 HSP70N组产生的IFNγ量比重组前高 (P <0 0 1) ,其余重组组产生的IFNγ量及所有重组组产生的IL 2均较重组前低 (P <0 0 1) ,而各重组组产生的抗体水平较重组前低 (P <0 0 5 )或无明显差异 (P >0 0 5 )。结论 :以HPV16E6 /E7为基础的DNA疫苗能增强小鼠的细胞免疫反应 ,对体液免疫几乎无影响。HPV16E6 /E7与结核杆菌HSP70N端重组后的疫苗与重组前比较不增强细胞免疫反应 ,不影响体液免疫反应。     

含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1-E7AS的构建及鉴定

目的:构建含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1- E7AS并鉴定,探讨腺相关病毒介导的反义E7基因技术用于治疗早期宫颈癌的可能性。方法:使用RT -PCR法扩增全长HPV16型E7基因,利用基因重组法将目的片段反向插入腺相关病毒骨架质粒pUF1并酶切鉴定。结果:RT PCR法扩增全长315bp的HPV16型E7基因,装入pGEM -Teasy载体进行测序,经NCBI数据库blast检索为100%的符合,经基因重组获得含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS,AccⅠ和KpnⅠ双酶切鉴定pUF1 E7AS。结论:含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS可成功构建。     

全酿酒酵母HPV16-E7疫苗的构建及免疫原性研究

目的构建人乳头状瘤病毒16型（HPV16）E7重组全酿酒酵母疫苗,评价疫苗的免疫效应。方法将HPV16 E7 cDNA亚克隆人酵母表达载体pYES2／NT,重组质粒转化酿酒酵母,经诱导表达、热灭活制备全酵母HPV16-E7疫苗,免疫C57BL／6小鼠。ELISA、MTT、LDH法分别检测疫苗诱导的细胞因子、脾淋巴细胞增殖和细胞毒性T淋巴细胞（CTL）应答水平。结果全酵母疫苗能够有效表达HPV16-E7蛋白;疫苗小鼠免疫,能够刺激脾淋巴细胞增殖,并诱导出Th1型细胞因子和特异性CTL杀伤效应;免疫效果优于单纯HPV16-E7蛋白免疫（P〈0．01）。结论全酵母疫苗既能表达HPV16-E7蛋白,又能有效诱导特异性CTL免疫应答。研究结果为进一步进行疫苗抗肿瘤疗效研究,提供了实验基础。     

人乳头状瘤病毒16型E7基因重组腺相关病毒载体的构建及E7 mRNA和蛋白在真核细胞中的表达

目的构建含人乳头状瘤病毒16型E7(HPV16E7)基因的重组腺相关病毒(rAAV)载体,并验证HPV16E7mRNA和蛋白在真核细胞中的表达。方法将含腺相关病毒(AAV)末端反向重复序列及HPV16E7基因的重组质粒pAAVMCSE7、含rep/cap基因的质粒pAAVRC及辅助质粒pAAVhelper共同转染胚胎肾细胞系HEK293细胞,回收、纯化病毒颗粒并鉴定,斑点杂交法测定病毒滴度,RTPCR技术、蛋白印迹法验证HPV16E7mRNA和蛋白在真核细胞中的表达。结果电镜鉴定结果显示真核细胞中有病毒颗粒存在,斑点杂交法测定病毒滴度为1×1011/ml。含HPV16E7基因的rAAV载体转染真核细胞后,在细胞内可检测到HPV16E7mRNA和蛋白的表达。结论本实验成功构建了含HPV16E7基因的rAAV载体,经验证HPV16E7mRNA和蛋白在真核细胞内有表达。     

腺病毒伴随病毒表达载体表达人乳头状瘤病毒16型E6/E7基因的构建及应用

目的　为了了解人乳头状瘤病毒 (Humanpapillomavirus ,HPV) 1 6型的E6 /E7基因在细胞恶性转化中所起的作用 ,利用腺病毒伴随病毒载体 (AAVHelper -FreeSystem)构建和表达人乳头状瘤病毒 1 6型E6 /E7基因。方法　在pLXSN1 6E6E7质粒中经PCR扩增回收HPV 1 6E6E7基因片段 ,连接于T载体上进行测序 ,将正确的HPV 1 6E6E7插入pAAV -IRES -hrGFP质粒 ,协同pAAV -RC质粒和pHelper质粒共转染HEK 2 93细胞 ,包装表达HPV 1 6E6E7基因的重组腺病毒伴随病毒 ,收获病毒 ,并检测病毒的感染效率。结果　在包装细胞系HEK 2 93细胞中能形成较高感染效率的腺病毒伴随病毒 ,激光共聚焦检测可发现HEK 2 93细胞内有绿色荧光蛋白表达 ,HEK 2 93细胞经PCR可扩增出特异性的HPV 1 6E6E7基因片段 ,经流式细胞仪检测重组病毒的感染效率为71 3%。结论　携带人乳头状瘤病毒 1 6型E6E7基因的腺病毒伴随病毒可感染细胞 ,并在细胞内表达 ,可望用于宫颈癌病因学的研究     

HPV58型E2蛋白表达纯化与多克隆抗体制备

目的:表达纯化重组人乳头瘤病毒(human papillomavirus,HPV)58型E2蛋白,制备多克隆抗体。方法:构建重组质粒p ET-28b-E2,转化至BL21(DE3)p Lys S中诱导表达,包涵体洗涤后经镍柱亲和层析分离得到纯化目的蛋白。用纯化的重组E2蛋白免疫新西兰白兔,制备兔抗HPV58型E2蛋白多克隆抗体,ELISA分析多克隆抗体的效价,免疫印迹检测抗体的特异性。结果:表达纯化了重组HPV58型E2蛋白,制备了高滴度和高特异性的多克隆抗体。结论:制备的多克隆抗体可用于对HPV58型E2蛋白进行精细B细胞线性表位鉴定。     

反义HPV16 E6/E7-EGFP重组质粒的构建及其对宫颈癌SiHa细胞的促凋亡作用

目的:构建HPV16早期基因E6/E7的反义重组质粒,探讨其对SiHa细胞的促凋亡作用。方法:将HPV16E6/E7基因片段反向克隆于真核表达载体pEGFP-C1并转染SiHa细胞,用RT-PCR方法检测转染后SiHa细胞E6、E7基因mRNA的表达,West-ernblot方法检测转染后E6/E7蛋白的表达,流式细胞仪检测转染后细胞的凋亡率。结果:成功构建携带HPV16E6/E7基因反义片段的真核表达载体,转染该质粒后,SiHa细胞E6、E7基因的mRNA和蛋白均明显下调;转染后细胞凋亡率为(59.3±11.3)%,明显高于转染空载体组[(9.4±1.8)%]和未转染组[(2.1±0.4)%](P<0.05)。结论:反义HPV16E6/E7基因可下调宫颈癌细胞中E6/E7癌基因的表达,诱导宫颈癌细胞凋亡,为宫颈癌的基因治疗提供了实验依据。     

抗HPV-16E_6单抗对宫颈癌靶向性的研究

目的 :评价抗HPV - 16E6 单克隆抗体靶向定位于宫颈癌组织的能力。方法 :用99Tcm 标记抗HPV - 16E6 单克隆抗体 (抗HPV - 16E6 +McAb ,实验组 )和正常鼠免疫球蛋白 (nMIgG ,对照组 )后 ,分别进行荷HPV - 16E6 +U14宫颈癌小鼠放射免疫显像 ,并比较放免结果。结果 :99Tcm 抗HPV - 16E6 McAb及99Tcm-nMIgG标记率分别为 91.97%和92 .4 9% ;标记前后抗体的免疫活性未改变 ;2 4h显像可见实验组肿瘤部位放射性浓集 ,图象清晰 ,肿瘤和非肿瘤组织放射性比值 (T/NT)明显高于对照组 (P <0 .0 5 )。结论 :抗HPV -16E6 单克隆抗体具有特异性定位于宫颈癌组织的能力 ,可能成为宫颈癌转移灶及复发性癌放免诊断和导向治疗的载体     

基于抗原表位分析的人乳头瘤病毒16L1壳蛋白基因克隆及表达纯化

目的:选择人乳头瘤病毒(HPV)16L1抗原性最强的一段抗原表位编码区,构建原核表达载体并利用大肠杆菌表达抗原蛋白,为HPV疫苗的研究及宫颈癌的诊断奠定基础。方法:借助DNAstar软件分析HPV16L1的CDS区,选择抗原性最强的一段抗原表位编码区,提取HPV16L1编码区的DNA。目的 DNA和p ET32a质粒分别进行双酶切,连接并转化DH5α大肠杆菌,筛选阳性克隆后转化BL21大肠杆菌,诱导产生HPV16L1重组蛋白,通过市售抗体对重组蛋白的抗原特异性进行鉴定。结果:利用DNAstar软件,选择了抗原表位较富集的碱基序列919~1314bp区段,按常规制备重组蛋白的方法成功制备HPV16L1重组蛋白。抗体鉴定表明,制备的重组蛋白抗原特异性良好。结论:有针对性地制备HPV16L1重组蛋白,可避免其他抗原表位的干扰。经鉴定,HPV16L1重组蛋白抗原特异性良好,将为HPV疫苗的研究及宫颈癌的诊断奠定基础。     

人乳头瘤病毒感染的致癌机制

人乳头瘤病毒 (HPV)感染是诱发宫颈癌的首要启动因素 ,90 %以上的宫颈癌组织携带高危HPVDNA ,其中HPV16型占6 0 %以上。HPV编码 6～ 8个早期蛋白 (E1～E8)和 2个晚期蛋白 (L1～L2 ) ,其中E5、E6和E7是其致癌的关键。E5蛋白 :HPV16E5蛋白分布于高尔基体、内质网和细胞膜上 ,能与多种细胞生长因子受体结合刺激细胞增殖 ,如表皮生长因子受体 (EGF R)、血小板生长因子受体 (PDGF Rβ)和集落刺激因子I受体 (CSF 1R)。E5蛋白可使主要组织相容性复合物I(MHC I)分子滞留于高尔基体 ,从而干扰病毒抗原的递呈 ,是HPV建立持续性感…     

IgG antibodies against human papillomavirus type 16 E7 proteins in cervicovaginal washing fluid from patients with cervical neoplasia

Abstract. Tjiong MY, ter Schegget J, Tjong-a-Hung SP, Out TA, van der Vange N, Burger MPM, Struyk L. IgG antibodies against human papillomavirus type 16 E7 proteins in cervicovaginal washing fluid from patients with cervical neoplasia.
Little information is available about the cervicovaginal mucosal antibodies against human papillomavirus (HPV) proteins. In this study specific IgG antibodies against HPV 16 E7 protein were determined in paired samples of cervicovaginal washing fluid and serum from patients with cervical cancer ( n = 22), cervical intraepithelial neoplasia (CIN) ( n = 38), healthy individuals ( n = 22), and serum from children ( n = 41) by a radioactive immunoprecipitation assay (RIPA). HPV 16 E7 specific IgG antibodies were found in cervicovaginal washings ( n = 8) and in sera ( n = 8) of the patients with cervical cancer. About 60% of the patients with HPV 16 positive cervical cancer had HPV 16 E7 specific IgG antibodies. Titration studies showed that the IgG antibody reactivity in cervicovaginal washings was higher than in the paired serum samples of six patients with cervical cancer ( P < 0.001). In the CIN group we found no IgG reactivity in the serum, but in five patients we found a low IgG reactivity in the cervicovaginal washings. No IgG reactivity was found in cervicovaginal washings and sera from healthy individuals and sera from children. HPV 16 E7 specific IgG antibodies seem to be locally produced in a number of patients with HPV 16 positive (pre)malignant cervical lesions. For more definitive evidence for the local production of these antibodies immunostaining should be performed to demonstrate the presence of specific anti-HPV 16 E7 IgG producing plasma cells in the cervical epithelium.     

Efficacy of human papillomavirus-16 vaccine to prevent cervical intraepithelial neoplasia: a randomized controlled trial

OBJECTIVE: Human papillomavirus (HPV) virus-like particle (VLP) vaccines have demonstrated effectiveness in preventing persistent HPV infections. Whether protection lasts longer than 18 months and, thus, impacts rates of cervical intraepithelial neoplasia (CIN) 2-3 has not yet been established. We present results from an HPV16 L1 VLP vaccine trial through 48 months. METHODS: A total of 2,391 women, aged 16-23 years, participated in a randomized, double-blind, placebo-controlled trial. Either 40 mug HPV16 L1 VLP vaccine or placebo was given intramuscularly at day 1, month 2, and month 6. Genital samples for HPV16 DNA and Pap tests were obtained at day 1, month 7, and then 6-monthly through month 48. Colposcopy and cervical biopsies were performed if clinically indicated and at study exit. Serum HPV16 antibody titer was measured by radioimmunoassay. RESULTS: Among 750 placebo recipients in the per protocol population, 12 women developed HPV16-related CIN2-3 (6 CIN2 and 6 CIN3). Among 755 vaccine recipients, there were no cases (vaccine efficacy 100%, 95% confidence interval [CI] 65-100%). There were 111 cases of persistent HPV16 infection in placebo recipients and 7 cases in vaccine recipients (vaccine efficacy 94%, 95% CI 88-98%). After immunization, HPV16 serum antibody geometric mean titers peaked at month 7 (1,519 milli-Merck units [mMU]/mL), declined through month 18 (202 mMU/mL), and remained relatively stable between month 30 and month 48 (128-150 mMU/mL). CONCLUSION: The vaccine HPV16 L1 VLP provides high-level protection against persistent HPV16 infection and HPV16-related CIN2-3 for at least 3.5 years after immunization. Administration of L1 VLP vaccines targeting HPV16 is likely to reduce risk for cervical cancer. LEVEL OF EVIDENCE: I.     

Investigation of uterine arterial chemoembolization and uterine arterial infusion chemotherapy for advanced cervical cancer before radical radiotherapy: a long-term follow-up study

Purpose

To evaluate the immunotherapeutic potentials for human dendritic cells (DCs) loaded with different HPV16-associated antigens, including HPV16E7 (E) protein, HPV16E7 polypeptide (P), as well as CpG-oligodeoxynucleotide (ODN) 2006 as a promising immune adjuvant for vaccination against cervical carcinoma.

Methods

DCs derived from human peripheral blood and cord blood were isolated and loaded with HPV-derived protein or peptides, in combination with CpG-ODN2006 as a potential adjuvant. The IL-12 level, the allogeneic T cell-stimulatory capacity and the cytotoxicity of cytotoxic T lymphocytes (CTLs) were evaluated in vitro. Furthermore, an immune reconstitution model of human cervical carcinoma in SCID mice was used to assess the anti-tumor effects in vivo. The tumor sizes, the expression of IgG and IFN-γ, and the presence of the human CD3⁺, CD4⁺ and CD8⁺ T cells were measured in the mice inoculated with different DCs.

Results

The antigen-loaded DCs displayed obvious anti-tumor activities in vitro and in vivo, and showed no toxicity to normal cells. The level of IL-12, an important cytokine for immune response, was up-regulated in all mice inoculated with antigen-loaded DCs. Stimulation and activity of CTLs were increased after treatment with antigen-loaded DCs. Significantly, DCs loaded with HPV16E7 polypeptide (P) showed the most distinguished immunotherapeutic activities, and such effect was further enhanced when HPV16E7 polypeptide (P) was used in combination with CpG-ODN2006. Interestingly, the same results were obtained in vivo: the tumor size was decreased, and IgG and IFN-γ levels were increased after the SCID mice were inoculated with the loaded DCs.

Conclusions

HPV16E7 polypeptide combined with the immune adjuvant CpG-ODN2006 could be a suitable HPV16-associated tumor antigen. The research provides a new strategy for generating DCs vaccines for immunotherapy of cervical cancer.     

Effects on fertility of immunizing mice with anti-idiotypic antibodies to porcine zona pellucida antigen

OBJECTIVE: To evaluate the effects on fertility by immunization with anti-idiotypic antibodies to porcine zona pellucida (PZP) antigen. METHOD: Anti-idiotypic antibodies (Ab(2)) were produced in New Zealand rabbits immunized with 17D3 monoclonal antibodies (mAbs) (IgG, Ab(1)) to PZP antigen. The antisera were first passed through immuno-affinity chromatography column linked to normal mouse IgG so as to remove the antibody bound to normal mouse IgG The passing elute was then purified by immuno-affinity chromatography using 17D3 mAbs to get the Ab(2). Female BALB/c mice, 5-week-old, were grouped and immunized with the Ab(2), PZP antigen, target antigen of the Ab(1) and normal rabbit IgG, respectively. The treated female mice were mated with male BALB/c mice and sacrificed at gestation day 10. Analyses included ELISA measurement of anti-ZP antibody titer, fetal number determination and evaluation of ovarian histomorphology. RESULTS: The Ab(2) appeared as a single protein band by SDS-PAGE. Shown by a competitive inhibition ELISA, the Ab(2) specifically bound to the variable region of the 17D3 Ab(1). Compared with controls, the female mice immunized with Ab(2) showed a decreased pregnancy rate and a statistically significant reduction in fetal numbers. Histological examination of ovaries demonstrated that Ab(2) exposure interfered less with follicular development than did exposure to PZP. CONCLUSION: Immunization of female mice with Ab(2) to PZP mAbs suppresses fertility and fetal numbers with minimal ovarian pathology.     

Human papillomavirus type 16 E7 peptide(38-61) linked with an immunoglobulin G fragment provides protective immunity in mice

OBJECTIVE: To explore whether the recombinant protein (Human papillomavirus (HPV) type16 E7 peptide(38-61) linked with an immunoglobulin G fragment) will generate protective immunity in mouse model. METHODS: In our study, we combined the HPV16 E7 peptide(38-61) with a murine IgG heavy chain constant region to construct a chimeric protein compound, which was highly expressed as inclusion bodies in a bacterial expression system with Escherichia coli. The purified chimeric protein was injected into C57BL/6 mice and the efficiency of the chimeric vaccine candidate was evaluated by antibody response assay, T cell proliferation assay, CTL assay, tumor challenge assay and therapeutic experiment. RESULTS: The chimeric vaccine candidate was able to induce anti-HPV antibodies as well as to elicit HPV16 E7-specific CTLs and T cell proliferation in a pre-clinical mouse model. It was also able to effectively protect mice against the challenge of HPV16-positive tumor cells, and to eradicate HPV16-expressing tumors in mice. CONCLUSIONS: The chimeric protein vaccine can induce E7-specific immune responses and protect mice against challenge of HPV16-positive tumor, even eradicate developed tumor. The results indicated a possibility to use the chimeric protein vaccine to protect human against HPV infection.     

HPV16E7肽疫苗对人免疫重建荷人宫颈癌细胞株SiHa细胞SCID鼠免疫功能的影响

目的:观察HPV16E7多肽疫苗对人免疫重建荷人宫颈癌细胞株SiHa细胞严重联合免疫缺陷(SCID)鼠移植瘤的影响。方法:对SCID鼠腹腔注射人外周血淋巴细胞(PBL)后24小时,皮下接种HPV16阳性的人宫颈癌细胞株SiHa细胞,建立人免疫重建荷SiHa细胞的SCID鼠模型,当移植瘤最小体积达100mm3时,三次背部皮下接种疫苗,每次接种间隔二周,观察荷瘤鼠一般生物学特性,检测血浆中人IgG含量、外周血中人CD3+、CD4+、CD8+T细胞、脾重、肿瘤浸润淋巴细胞(TIL)以及脾细胞毒杀伤功能。实验设立空白组,仅为腹腔内注射PBL,疫苗成份仅为不完全佐剂(IFA);随机多肽组,为腹腔内注射PBL,皮下接种SiHa细胞,疫苗成份仅为随机多肽+T辅助多肽+IFA;T辅助多肽组,为腹腔内注射PBL,皮下接种SiHa细胞,疫苗成份仅为T辅助多肽+IFA;多肽治疗组,为腹腔内注射PBL,皮下接种SiHa细胞,疫苗成份为HPV16E711-20(YMLDLQ-PETT),HPV16E786-93(TLGIVCPI)+T辅助多肽+IFA。结果:各组SCID鼠血浆中人IgG水平,8周内随重建时间延长而升高(P<0.05),多肽治疗组达2957.85μg/m l,但各组间无显著性差异;外周血中均可检测到人CD3+、CD4+、CD8+T细胞,但各组间无显著性差异(P>0.05);与空白组比较,其他三组SCID鼠脾重均显著增加(P<0.05)。组织学观察到移植瘤局部TIL浸润及明显的出血和坏死,但经免疫组化未检测到人CD8+T细胞。与空白组比较,多肽治疗组、随机多肽组、辅助多肽组的脾细胞毒杀伤功能均显著降低(P<0.05)。结论:人免疫功能重建的荷SiHa细胞的SCID鼠体内重建的人免疫功能随荷瘤时间的延长受到抑制,多肽疫苗在一定程度上能减轻免疫抑制状态。     

人乳头瘤病毒16/18型与宫颈癌

人乳头瘤病毒（HPV）16/18型和宫颈癌的关系密切,在宫颈癌发生发展中起重要作用。HPV16最易导致宫颈鳞癌,HPV18最易导致宫颈腺癌。HPV16/18致癌机制主要是通过病毒基因组中致癌蛋白E6,E7与抑癌基因p53和pRb结合,E6抑制p53的活性,E7灭活Rb基因的活性,致肿瘤发生。常用的HPV检测方法有原位杂交法（ISH）、第二代杂交捕获试验（HCⅡ）、聚合酶链反应（PCR）等。针对HPV16/18的预防和治疗性疫苗成为研究热点。综述HPV16/18型和宫颈癌关系研究进展。     

含编码精子膜肽段cDNA的重组沙门氏菌和痘苗病毒粘膜免疫大鼠所致免疫效应及抗生育效应的初步研究

应用合编码精子膜肽段YAL－198cDNA片段的重组减毒沙门氏菌（NonpathogenicSalmonelladublin）和重组痘苗病毒天坛病毒株（TianTanstrain）对雌性大鼠通过口服、呼吸道喷涂、阴道滴注等途径进行粘膜免疫。在大鼠血清和阴道冲洗液中均得到了相应的抗体效价。通过抗生育实验,实验组5只大鼠中得到较高的（60～80％）抗生育率。验证了粘膜免疫的可行性和有效性。从血清和阴道冲洗液中的抗体效价来看,特别是在重组沙门氏菌免疫组中,阴道冲洗液中的抗体效价与大鼠抗生育率之间里正相关,通过3次合笼表明,大鼠的生育能力可逐步恢复,并在初次免疫10个月后基本恢复正常。从而证明了两种抗生育疫苗抗生育效应的可逆性。     

卵巢癌抗独特型抗体6B11特异性表位肽在体内诱导抗原特异性体液免疫应答

目的探讨卵巢癌抗独特型抗体6B11表位多肽与诱导免疫应答之间的关系。方法采用化学合成的方法，合成抗独特型抗体6B11轻重链可变区的六条CDR区，分别以三种不同浓度免疫BALB／c小鼠。采用ELISA的方法检测免疫鼠血清，进行体内免疫学功能研究。结果6B11抗独特型抗体的六条CDR肽分别加入佐剂免疫小鼠后，其中有两条肽可以诱导出Ab3，分别为重链的CDR2区和轻链的CDR2区。结论这两条肽作为后选肽有可能是6B11抗独特型抗体特异性的CTL表位。