PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16 L1/E6-E7 CHIMERIC RECOMBINANT DNA VACCINE

Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.

Transient over-expression of human papillomavirus type 16 E6 protein down-regulate the secretion of TNF-αor IL-1β LPS-induced from macrophages

In order to provide the experimental basis for the further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16 （HPV16）, the eukaryotic expression vector pcDNA3.1 （-）/E6 was used for the study on the effect of E6 protein to influence the secretory activity of LPS-induced 3MP-1-macrophages, and the reconstructed plasmid pcDNA3.1 （-）/E6 was transfected into THP-1-macrophages. The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-α or IL-1β was examined by ELISA. All of data were analyzed by SPSS12.0. As demonstrated by Western blot analysis, the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1 （-）/E6 in THP-1-macrophages could be detected. However, as demonstrated by ELISA assay, the level of TNF-α or IL-1β in lysates of THP-1-macrophages showed an obvious difference between the pcDNA3.1 （-）/E6 group and the LPS control group or the pcDNA3.1 （-） control group （P 〈 0.01）, but no significant difference existed between pcDNA3.1 （-） control group and LPS control group （ P 〉 0.05）. All these results illustrate that the transient over-expression of HPV6 E6 protein reduces the production of TNF-α and IL-1β induced by LPS in THP-1-macrophages.

HUMAN PAPILLOMAVIRUS TYPE 16 L1 PROTEIN CAN BE EXPRESSED IN LIVE ATTENUATED SHIGELLA FLEXNERI 5A STRAIN SH42

Objective Attenuated strains of Shigella are attractive live vaccine candidates for eliciting mucosal immune responses which is a suitable carrier for the prophylactic human papillomaviruses (HPV) vaccine development, To examine the potential of a live Shigella based prophylactic HPV vaccine, HPV16L1should be expressed in attenuated shigella strain. Methods A Shigella large invasive plasmid (icsA/virG) based prokaryotic expression plasmid pHS3199 was constructed. HPV16L1 gene was inserted into plasmid pHS3199 to form pHS3199-HPV16 L1 construct, and pHS3199-hpv16L1 was electroporated into a live attenuated shigella strain sh42. The expression of HPV16L1 protein was demonstrated by Western blotting with monoclonal antibody to HPV16L1, The genetic stability of recombinant strain sh42-HPV16 L1 was monitored by consecutive passage culture. Invasive ability of sh42-HPV16L1 was evaluated by Hela cell infection assay. Results HPV16 L1 protein can be expressed in recombinant strain sh42-HPV16 L1, and the protein stably expressed over 140 generations. The invasive ability of sh42-HPV16L1 was diminished dramatically compared to its parent strain, but not abolished completely. Conclusion HPV16L1 protein was constitutively expressed in the attenuated strain of shigella flexneri sh42, and maintained partial invasive ability. Our strategy may represent a promising vaccine candidate against genital HPV16 infection.

DETECTION OF HUMAN PAPILLOMAVIRUS TYPES 16, 18 DNA RELATED SEQUENCES IN BRONCHOGENIC CARCINOMA BY POLYMERASE CHAIN REACTION

In studying the relationship between human papillomavirus (HPV) and bronchogenic carcinoma, 'high-risk' HPV 16, 18 DNA sequences were detected in samples from 50 lung cancer patients, 18 patients with benign pulmonary diseases and 4 fetal lung tissues by polymerase chain reaction (PCR) and dot-blot hybridization with biotin-labelled probes. The results showed that HPV 16, 18 DNA related sequences were found in 32% of lung cancer specimens, with 10 cases of HPV 16, 5 cases of HPV 18 and 1 case of both types. 48.15% (13 / 27) of squamous cell carcinomas were shown to be positive for HPV 16, 18 DNA. In addition, two adenocarcinomas and one small cell carcinoma were positive for HPV 16 DNA. No specimens from benign diseases tissues and fetal lung tissues showed positive results. These results suggest that primary bronchogenic carcinoma is related to HPV infection.

Polymorphism in the upstream regulatory region of human papilloma virus type 16 from the cervical cancer biopsies in Xinjiang Uygur women

To investigate the mutations in the upstream regulatory region (URR) of human papillomavirus type 16 (HPV-16) from the cervical cancer biopsies in Xinjiang Uygur women and its relationship to the high incidence of cervical cancer in the southern Xinjiang, the tissue DNA was extracted from the cervical cancer biopsies, and the URR segment of HPV-16 DNA was amplified, sequenced and analyzed. Thereafter, the polymorphism of URR in HPV-16 was then analyzed. It was demonstrated that the positive rate detected for the presence of URR in HPV-16 was 89.47% (17/19). Compared with the previously published sequence in URR of prototype HPV-16, some mutations were detected in the sequence of URR. The mutations in 17 URR fragments of HPV-16 could be divided into 11 patterns (XJU-1 to XJU-11) at nucleic acid level, in which each of XJU-1 and XJU-4 accounted for 23.53% (4/17), and other patterns of mutation accounted for 5.88% (1/17) . In comparison with the URR of prototype HPV-16, the DNA identity of these patterns was 98.50%-99.68% . In these 17 URR fragments, two point mutations occurred at position 7192 (G to T) and position 7520 (G to A) and they appeared to be constant in Xinjiang area. These two mutations were ubiquitous in the Asia-American type and conferred strong infection activity and carcinogenicity of this virus. In addition, the mutations at position 7729 (A to C), position 7843 (A to G) and position 7792 (C to T) could enhance its transcription activity considerably. It is concluded that some mutations occur in URR gene of HPV-16 in the cervical cancer biopsies taken from Uygur women in Xinjiang area, suggesting that certain relationship exists among the mutations in URR of HPV-16, the phylogeny of HPV-16 and the high incidence of cervical cancer in southern part of Xinjiang area.

重庆永川地区妇科门诊人乳头瘤病毒亚型分布的研究

目的:了解23种人乳头瘤病毒(Human papillo maviruses,HPV)型别在永川地区人群中的感染情况及分布特点,为本地区人乳头瘤病毒感染诊断及其相关疾病的防治提供重要依据。方法:应用基于反向膜杂交的基因芯片技术对665例临床送检标本进行分型检测,所得数据用SPSS等软件进行统计分析。结果:HPV总感染率为37.65%,23种基因型检出20种,其中单一型别感染率为27.25%,两型混合感染率为7.22%,3型(包括3型)以上混合感染率为3.18%。HPV亚型分布情况为:HPV16型9.92%、HPV6型5.74%、HPV11型4.36%、HPV33型1.32%、HPV58型1.05%、HPV43型1.02%、HPV18型0.75%,其余型别3.29%;高危型HPV在≤20、21～30、31～40、>40年龄组的感染率依次为14.1%、15.2%、17.2%、17.3%(χ2=3.0,P>0.05),组间比较差异无显著性;低危型HPV在各年龄组的感染率依次为20.3%、11.7%、10.1%、10.1%(χ2=23.28,P<0.01),组间比较差异具有显著性。结论:本地区人群HPV感染率较高,且以单一亚型感染为主,其中HPV16为高危型HPV感染主要的型别,低危型HPV感染以HPV6和HPV11为主;HPV各型别的感染率与年龄有一定的关系,该结果为防治宫颈癌、尖锐湿疣等其它HPV相关疾病提供了理论依据。

云南省妇科门诊女性HPV检测结果分析

目的了解21种人乳头瘤病毒(human papillomavirus,HPV)型别在云南省妇科门诊人群中的感染情况及型别分布和年龄分布等特点.方法应用基于膜杂交的低密度基因芯片技术对2 221例标本进行分型检测,所得数据用SPSS软件进行统计分析.结果 HPV总感染率为20.76%,高危型HPV感染率18.21%.21种基因型全部检出,其中单一型别感染率为16.21%,2型混合感染率为3.60%,3型(包括3型)以上多重感染率为0.95%.HPV亚型感染率最高的分别是HPV52型4.73%,HPV16型4.05%,HPV58型2.70%,CP8304型2.39%,HPV33型1.53%;各年龄组HPV阳性率组间比较差异无显著性(χ2=9.448,P=0.092).结论本地区人群HPV感染率较高,且以单一亚型感染为主,其中HPV52为高危型HPV感染最主要的型别,低危型HPV感染以CP8304型为主;HPV感染是各年龄段女性共同的致病因素.

11625名长沙市女性人乳头瘤病毒检测结果分析

目的了解人乳头瘤病毒(HPV)基因亚型在长沙市女性人群中的感染情况、型别分布和年龄分布等特点。方法选择2012年9月至2013年6月中南大学湘雅三医院体检的长沙市女性共11 625例,应用导流杂交基因芯片技术对其进行分型检测,分析HPV感染率及感染亚型。结果 HPV总感染率为14.8%,HPV亚型感染率从高到低依次为52型(4.0%),58型(2.4%),CP8304型(2.1%),16型(2.0%),53型(1.7%)。其中单一型别感染率为11.9%,2型混合感染率为2.4%,3型(包括3型)以上多亚型感染率为0.5%。不同年龄段HPV感染率比较,差异有统计学意义(χ2=16.48,P<0.01),随着年龄的增加,HPV的感染率逐渐增加。结论长沙市女性人群中HPV感染率不高,50～60岁的感染率稍高。以单一亚型感染为主,其中HPV52为高危型HPV。

Enhanced immunization after intranasal coadministration of Escherichia coli heat-labile enterotoxin B subunit and human papillomavirus 16-L1 DNA vaccine

Human papillomavirus （HPV）, mainly types 16 and 18, are the most important initiating agents of cervical cancer. Prevention of high-risk HPV infections is a potentially effective approach to control HPV associated cervical cancer.

HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

Using human papillomavirus type 16 （HPV16） E7 as an antigen and Heat Shock Protein 70 as adjuvant, we constructed a DNA vaccine by linking HSP70 gene to E7^C91G; gene. Mice, after being immunized with E7^C91G;-HSP70, E7^C91G/HSP70, E7^C91G, and wild E7 DNA vaccines respectively, produced E7 specific CD8^＋ T-cell precursor frequencies of 280.33±2.52, 144.34±4.04, 164.34±5.13 and 82.33±3.51 respectively within every 1 × 10^5 mouse splenocytes. This proves that E7^C91G-HSP70 fusion vaccine can significantly enhance the E7 specific cellular immunity within the mice body（p〈0. 01）. After being immunized with E7^C91G-HSP70 fusion vaccine, tumor-bearing mice of the group being treated have significantly longer latency and survival periods, comparing with other three categories of E7 vaccines. Experiment shows that this vaccine has a significant effect on enhancing E7 positive tumor-treatment within mice body. After being immunized with E7^C91G-HSP70 vaccine, there were no pathological changes found in livers, kidneys and spleens of the mice, which proves that the vaccine is quite safe. After all, E7^C91G-HSP70 fusion vaccine has a much stronger tumor- treatment effect than thai of wild type E7 DNA vaccine.

THE CONSTRUCTION OF HPV16-L1 RECOMBINANT PLASMID AND ITS EXPRESSION IN MAMMALIAN CELLS

Infection with high risk human papillomavirus is regarded as the major risk factor in the development of cervical cancer. In this study, HPV16 L1 eukaryotic expression plasmids pcDNA LI were constructed, which were transfected into mammalian cells Cos 7. The expression of HPV16 L1 in transfected cells were identified by in situ hybridization, immunospot and immunocytochemistry. HPV16 L1 mRNA transcription and L1 protein expression were found in recombinant plasmid transfected cells. This expression system will provide us with plentiful resource for HPV16 L1 immunological study and will be helpful for the design of HPV16 prophylactic vaccine.

Cloning of HPV16 E2 Gene from a Biopsied Cervical Cancer Sample

To clone HPV16 E2 gene from a biopsied cervical cancer sample Materials & Methods HPV16 E2 gene was amplified from specimen derived from a HPV 16 positive patient, then cloned and sequenced. Results The full length of HPV 16 E2 gene was successfully cloned. In comparison with the prototype accepted by GenBank, six point mutations in HPV 16 E2 nucleotide acid sequence were identified. Of them, three were missense, and one was in the overlapping E4 gene and was synonymous to E4. Conclusion HPV16 E2 gene was successfully cloned, and some nucleotide acids in its sequence were different from the prototype.

宫颈癌组织中人乳头状瘤病毒16型DNA序列的检测

应用DNA斑点分子杂交技术检测了54例宫颈癌组织中人乳头状瘤病毒16型DNA序列,并应用DNA Southern印迹杂交技术分析了部分斑点杂交阳性的标本。结果表明,50%的宫颈癌标本中HPV16DNA阳性,而在18例正常宫颈组织中未测到HPV16DNA序列;Southern印迹杂交发现HPV16DNA以整合状态存在。提示HPV16与宫颈癌的发生密切相关。

共表达HPV16型E6和E7突变基因重组痘苗病毒的构建及其抗肿瘤免疫效果的观察

目的 构建用于子宫颈癌治疗的HPV16型E6和E7重组痘苗病毒实验性疫苗株 ,并对其抗肿瘤免疫效果进行初步评价。方法 以痘苗病毒为载体、利用同源重组技术构建共表达HPV16E6和E7基因的重组痘苗病毒。该病毒免疫C57BL 6小鼠后 ,检测其免疫原性和抗移植瘤生长情况。结果 PCR结果显示 ,重组病毒VmE6E7的TK基因内插入了分别由痘苗病毒早晚期启动子H6和7.5K表达的ME6和ME7 1基因。动物实验结果表明 ,rVmE6E7在C57BL 6小鼠体内可诱发E6和E7特异性抗体产生 ,被免疫小鼠能够抵抗HPV16E6E7转化的同系肿瘤细胞的攻击。结论 获得 1株用于宫颈癌治疗的HPV16型实验疫苗株 ,为进一步研制人用HPV16型疫苗株奠定了基础。

Immune response to plasmid DNA encoding HPV16-L1 protein

Objective To test the immunogenicity of recombinant plasmid DNA containing human papillomavirus type 16 L1 (HPV16 L1) coding sequence of mice Methods The HPV16 L1 encoding sequence was generated by polymerase chain reaction (PCR), and inserted into TA cloning vector PCR Ⅱ, then cloned in the eukaryotic expression vector pcDNA3 1 with CMV promoter The recombinant plasmid DNA pcDNA L1 was transferred into Cos 7 cells and used to immunize BALB/c mice via muscular injection The expression of HPV16 L1 in transferred cells was identified by immunospot and immunocytochemistry, which tested specific anti HPV16 L1 antibody in the serum of immunized mice Results Using the immunospot technique, we found L1 protein expression in pcDNA L1 transferred cells The immunocytochemistry studies demonstrated that the L1 protein was located in nuclei In immunized mice, specific anti HPV16 L1 antibodies could be detected by immunospot and immunocytochemistry 28 days after the first immunization and last at least 41 days Conclusions We constructed HPV16 L1 eukaryotic expressing plasmid whose DNA could induce immuno humoral response in mice This observation will be helpful in designing HPV16 prophylactic vaccine