Detection of HPV Types and Neutralizing Antibodies in Women with Genital Warts in Tianjin City,China

The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV. The results revealed that 36% （18/50） of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560, of which 22%（11/50）, 12%（6/50）, 10%（5/50）, 4%（2/50）, 4%（2/50） and 2%（1/50） were against HPVs -6, -16, -18, -58, -45 and -1 l, respectively. Additionally, 60% （30/50） of samples were HPV DNA-positive, in which the most common types detected were HPV-68（18%）, HPV-16（14%）, HPV-58（12%）, HPV-33（8%） and HPV-6, HPV-11, HPV-18 and HPV-52 （6% each）. The concordance between HPV DNA and corresponding neutralizing antibodies was 56% （28/50） with a significant difference （P〈0.05）. The full-length sequences of five HPV types （HPV -42, -52, -53, -58 and -68） were determined and exhibited 98%-100% identities with their reported genomes. The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.

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.

人类乳头状瘤病毒与鼻息肉的关系

目的 探讨人类乳头状瘤病毒感染与鼻息肉发病的相关性。方法 取70例鼻息肉组织标本,用HPY-DNA-Typing方法进行人类乳头状瘤病毒(HPV-DNA)检测及病毒分型,24例正常鼻腔粘膜作对照。结果70例鼻息肉中有19例(27.14％)HPV-DNA感染,24例正常鼻腔粘膜中1例感染HPV-DNA(4.17％),HPV分型发现以HPV-5型为主。结论 鼻息肉发病可能与HPV感染有一定关系。

不同宫颈病变组织中人乳头瘤病毒16型E5基因的研究

目的:探讨人乳头瘤病毒(Human papillomavirus type,HPV)16型E5早期基因在临床宫颈炎、宫颈上皮内瘤变(Cervical intraepithelial neoplasia,CIN)Ⅰ、Ⅱ、Ⅲ型及宫颈癌患者中的存在情况,判断HPV16E5是否能作为临床宫颈癌的早期诊断治疗靶点,以利进一步研究HPV16的致癌机制。方法:从临床标本中筛选出HPV16阳性标本61例,根据病理诊断分为5组(宫颈炎组、CINⅠ级、CINⅡ级、CINⅢ级及宫颈癌组),再应用PCR的方法分别测定各组组织中HPV16E5DNA的表达情况。结果:宫颈炎组E5DNA阳性率为58.33%,CINⅠ级为63.64%,CINⅡ级为70.00%,CINⅢ级为44.44%,宫颈癌为77.78%,各组间阳性率差异无统计学意义。结论:E5DNA不仅仅在宫颈癌早期存在,因而E5不能作为临床宫颈癌的早期诊断靶点,但可作为宫颈癌疫苗研究的靶点。

The effect of human papilloma virus type 16 E7 protein expression on growth of RMA cells in vitro and in vivo

Objective： The aim of this study was to study the effect of human papilloma virus （HPV） type 16 E7 protein ex- pression on growth of RMA cells in vitro and in vivo. Methods： The recombination vector pcDNA3.1-E7 carrying wild type HPV 16 E7 was identified by sequencing. The recombination vector pcDNA3.1-E7 was transfected into mouse lymphadenoma cell line RMA by liposome, and the monoclonal cells transfected stably were obtained by antibiotics G418 sieving and limiting dilution assay. RT-PCR method was used to detect the expression of HPV 16 E7 mRNA in RMA-E7 cells. The growth of RMA cells and RMA-E7 cells cultured in vitro was tested by Cell Count Kit-8. RMA-E7 cells and RMA cells were subcutaneously inoculated in syngeneic mice respectively, the tumor size was measured by sliding caliper twice a week, and the E7 protein expression in tumor tissue of mice was detected by Western blot after tumor formation. The kinetics of cytolytic activity of E7 specific T cells in tumor-bearing mice was measured by LDH kit. Results： Sequencing of recombination vector showed the target gene which was inserted into the recombinant was correct, and RMA-E7 cells expressing E7 protein stably were obtained by limited dilution assay. There were no obvious differences in morphous and growth velocity between RMA cells and RMA-E7 cells in vitro. RMA-E7 cells grew in syngeneic mice were significantly slower than RMA cells. The E7 protein was ex- pressed stronger in RMA-E7 cells in vivo than in vitro. The cytolytic ability of ET-specific CTL was activated at the early stage, reached the maximum at the middle stage, and lost at the end stage. RMA-E7 cells isolated from the tumor-bearing mice were more resistant to E7-specific CTL killing than RMA-E7 cells cultured in vitro. Conclusion： The E7 protein expression has no obvious influence on growth of RMA-E7 cells in vitro, and can suppress growth of RMA-E7 cells in vivo. The activity curve of E7 specific CTL approximately presents ＂bell＂ shape. The RMA-E7 cells grew in vivo had a high expression levels of E7 protein, and more resistant to E7-specific CTL killing than those cultured in vitro. The E7 protein expression in vivo not only initiates immune activation, but also induces immune tolerance.

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.