HPVL1保守序列肽段诱导多型别HPV抗体的初步研究

为探索一段人类乳头瘤病毒(HPV)L1蛋白中长12个氨基酸残基的保守序列是否能诱导产生多型别HPV抗体,我们通过B细胞表位预测和多序列对比,筛选出一条HPV L1蛋白保守肽段,并人工合成此肽段,加弗氏佐剂后免疫家兔,对照组只用弗氏佐剂。先用ELISA的方法检测免疫家兔血清中抗体滴度;再用免疫细胞化学、免疫细胞荧光、Western blot和免疫组织化学的方法检测此抗血清与HPV阳性的宫颈癌细胞株和宫颈组织的反应情况。结果发现,用ELISA法检测抗血清效价在1∶25600以上,而免疫细胞化学、细胞免疫荧光、Western blot和免疫组织化学等方法均检测出抗血清能与多型别HPV进行反应,而对照组呈阴性。此结果表明,该多肽可诱导产生针对多型别HPV的抗体,对于研发制备HPV、HPV L1诊断试剂盒有重要意义。     

HPV L1 C-末端保守序列短肽诱导HPV多型别交叉抗体的初步研究

通过对HPV L1序列进行比对,发现HPV L1 C-末端存在长30个氨基酸残基的保守序列短肽;出于检测短肽是否可以诱生HPV多种型别交叉抗体的目的,将该序列短肽加弗氏佐剂用于日本大耳白兔和BALB/c小鼠免疫,然后用ELISA方法检测此免疫动物血清及其分泌物中的IgG抗体滴度,发现此免疫动物体内已诱生出高滴度的血清IgG抗体(>1∶20000);再用ELISA、免疫组织化学和Western blot的方法对此诱生血清抗体与HPV阳性宫颈癌细胞株的反应情况进行检测,发现这些短肽抗血清可与16、18型HPV L1很好地进行反应,其对照组呈现阴性。这一研究结果表明短肽可以诱生HPV多种型别交叉抗体。它对后续研发HPV L1广谱疫苗或检测试剂盒具有重要意义。     

含HPV16L1基因重组腺病毒和1型重组AAV载体联合免疫效果的研究

目的 对表达HPV16L1抗原的重组腺病毒及1型重组AAV载体联合免疫效果进行研究.方法 分别构建含密码子优化型HPVl6LI基因重组腺病毒rAd-mod.HPV16L1及1型重组从V载体rAAV1-mod-HPV 16L1,将纯化的重组AAV病毒载体以肌注及滴鼻途径单独及联合免疫C57BL/6小鼠,使用体外中和实验检测各组小鼠血清中特异性中和抗体.结果 rAAV1-med-HPV16L1单独及与rAd-mod-HPV16L1联合肌注可诱导高滴度的血清中和抗体,在初免后第16周抗体滴度显著高于其他免疫组,联合肌注组诱导的抗体滴度高于单独肌注组;重组病毒联合滴鼻虽能产生一定的免疫加强作用,但抗体滴度仍显著低于rAAV1-mod-HPV16L1单独及联合肌注组.结论 型重组从V载体联合重组腺病毒以初免.加强模式肌注可诱导更高滴度的血清中和抗体.     

表达HPV 16型结构蛋白的非复制型重组痘苗病毒的构建与鉴定

目的 构建表达人乳头瘤病毒16型(HPV16)结构蛋白的非复制型重组痘苗病毒人用疫苗株。方法 采用聚合酶链反应技术(PCR)扩增并克隆HPV16主要结构蛋白L1和次要结构蛋白L2基因;将经过结构修饰的L1和L2基因插入痘苗病毒表达载体,通过与痘苗病毒在宿主细胞中同源重组后,筛选共表达L1／L2蛋白的重组痘苗病毒并对其进行鉴定。结果 DNA序列分析证实PCR扩增所获L1和L2克隆基因是正确的;斑点杂交结果表明重组病毒基因组中有L1和L2基因插入;该重组病毒在人源细胞中不复制或低水平复制,但可稳定表达相对分子质量为57000的L1蛋白和相对分子质量为90000的L2蛋白。结论 获得1株稳定表达HPV16 L1／L2蛋白的非复制型重组痘苗病毒人用疫苗株。     

Sequence-based genotyping HPV L1 DNA and RNA transcripts in clinical specimens

We developed a direct sequence-based genotyping method to detect single and multiple HPV L1 DNA and RNA types in genital and dermatological specimens. Our method couples PCR amplification of a highly conserved HPV L1 segment using a broad spectrum-generic primer cocktail mix with automated sequencing of amplified PCR products, followed by GenBank sorting of sequencing data. We genotyped 5 skin and 30 cervical HPV DNA-positive specimens using this method and established its first experimentally derived working cutoff value with the aid of commercial hybridization-based techniques. We suggest that sequence-based genotyping of appropriately amplified DNA and RNA products may serve as a primary HPV detection method in dermatological specimens. It can be applied as an all-purpose genotyping method for rare HPV types not detectable by commercial hybridization-based techniques and for sorting multiple HPV infections by order of prevalence.     

Human papillomavirus (HPV) genotyping using paired exfoliated cervicovaginal cells and paraffin-embedded tissues to highlight difficulties in attributing HPV types to specific lesions

Defining type-specific human papillomavirus (HPV) infections within cervical tissues is important for understanding the pathogenesis of cervical neoplasia and assessing the effectiveness of prophylactic vaccines with limited type-specific spectra. We compared HPV DNA-testing results from 146 matched exfoliated-cell and formalin-fixed-tissue specimens collected by cervicovaginal lavage (CVL) within 90 days of each other from women with histologically confirmed cervical intraepithelial lesions (CIN). The CVL specimens were HPV typed using a MY09/11 L1 consensus primer PCR method followed by dot blot hybridization. The tissue specimens were HPV typed using an SPF(10) line probe assay HPV detection system. Of the 146 specimen pairs with evidence of CIN in the tissue, 91.8% were positive for one or more HPV types in both the tissue and cellular specimens. Tissue sections were more likely to be HPV negative (P < 0.01). Typing directly from tissue sections resolved multiple infections detected in exfoliated cells to a single HPV type in only 46.9% of cases. Combined use of both specimen types to attribute lesions to HPV type 16 (HPV-16) and/or -18 led to 43.1% attributed to HPV-16 and/or -18 by both specimen types and 19.9% attributed to HPV-16 and/or -18 by one, but not both, specimen types. Unambiguous attribution of cervical lesions to a single, specific HPV type remains a difficult proposition. Use of multiple specimen types or the development of highly sensitive and robust in situ hybridization HPV-testing methods to evaluate the certainty of attribution of lesions to HPV types might provide insights in future efforts, including HPV vaccine trials.     

Clinicopathological Implications of Human Papilloma Virus (HPV) L1 Capsid Protein Immunoreactivity in HPV16-Positive Cervical Cytology

Background: The objective of this study was to investigate the expression of human papilloma virus (HPV) L1 capsid protein in abnormal cervical cytology with HPV16 infection and analyze its association with cervical histopathology in Korean women.Material and Methods: We performed immunocytochemistry for HPV L1 in 475 abnormal cervical cytology samples from patients with HPV16 infections using the Cytoactiv^® HPV L1 screening set. We investigated the expression of HPV L1 in cervical cytology samples and compared it with the results of histopathological examination of surgical specimens.Results: Of a total of 475 cases, 188 (39.6%) were immunocytochemically positive and 287 (60.4%) negative for HPV L1. The immunocytochemical expression rates of HPV L1 in atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cancer were 21.8%, 59.7%, 19.1%, and 0.0%, respectively. LSIL exhibited the highest rate of HPV L1 positivity. Of a total of 475 cases, the multiple-type HPV infection rate, including HPV16, in HPV L1-negative cytology samples was 27.5%, which was significantly higher than that in HPV L1-positive cytology samples (p = 0.037). The absence of HPV L1 expression in ASCUS and LSIL was significantly associated with high-grade (≥cervical intraepithelial neoplasia [CIN] 2) than low-grade (≤CIN1) histopathology diagnoses (p < 0.05), but was not significantly different between HPV16 single and multiple-type HPV infections (p > 0.05). On the other hand, among 188 HPV L1-positive cases, 30.6% of multiple-type HPV infections showed high-grade histopathology diagnoses (≥CIN3), significantly higher than the percentage of HPV16 single infections (8.6%) (p = 0.0004)Conclusions: Our study demonstrates that the expression of HPV L1 is low in advanced dysplasia. Furthermore, the absence of HPV L1 in HPV16-positive low-grade cytology (i.e., ASCUS and LSIL) is strongly associated with high-grade histopathology diagnoses. The multiplicity of HPV infections may have an important role in high-grade histopathology diagnoses (≥CIN3) in HPV L1-positive cases.     

High frequency of multiple HPV types in cervical specimens from Danish women

Genital human papillomavirus infection (HPV) is common and usually harmless. However, chronic cervical infection with high‐risk HPV types can cause cell changes that may eventually lead to cancer. To determine the frequency of individual HPV types among mixed infections, we examined the type distribution among cervical specimens from more than 1000 Danish women. We also examined the HPV type distribution and the frequency of single and multiple HPV types for specimens from 113 women who underwent conization and were diagnosed with cervical intraepithelial neoplasia grade II or worse (CIN2+). Using microarray technology, we found that 49% of the HPV‐positive patients were infected with multiple HPV types. Among the CIN2+ diagnosed women, this frequency was 41%. The most frequently found high‐risk HPV type was HPV‐16, which was found in 25% of the HPV‐positive cervical specimens. Among the HPV positive CIN2+ diagnosed women, 48% were HPV‐16 positive. Women younger than 30 years of age had a higher frequency of multiple infections (61%) than women older than 30 years (39%). We conclude that cervical infection with multiple HPV types is common among women in all age groups and among women with or without the diagnosis of CIN2+.     

HPV16L1／L2蛋白单克隆抗体的制备

用真核表达的ＨＰＶ１６Ｌ１／Ｌ２蛋白作为抗原，经免疫、融合、选择性培养、克隆化等过程，我们建立了两株抗ＨＰＶ１６Ｌ１／Ｌ２蛋白的杂交瘤细胞株（另有数株仍在建株中）。免疫斑点法检测证明，它们产生的抗体，只与ＨＰＶ１６Ｌ１／Ｌ２蛋白起反应，而不与ＨＰＶ１６Ｅ６、Ｅ７蛋白、人血清蛋白和牛血清蛋白起反应。另外，免疫组化试验证明，这种抗体可应用于临床ＨＰＶ１６感染的检验，具有敏感、特异、准确、快捷、经济的优点。     

Detection of HPV 16 and HPV 18 DNA in the blood of patients with cervical cancer

Persistent infection of the uterine cervix with high-risk human papillomaviruses (HPV) is causally associated with cancer of the cervix. A few studies have reported the presence of HPV DNA in the blood of women with cervical neoplasia. The aim of this study was to determine if HPV DNA could be detected in whole blood of women with a range of cervical pathologies and with HPV 16 or 18 cervical infections and if there is a correlation between cervical lesion grade and the appearance of HPV DNA in the circulatory system. Forty-five women with histologically graded cervical cancer were confirmed to have cervical HPV 16 or 18 infections. Eleven (24.4%) of these women had detectable HPV 16 or 18 in their blood. The HPV types detected in the blood matched those detected at the cervix. No HPV 16 or 18 DNA was detected in the blood of 32 women with pre-cursor cervical lesions or normal cervical pathology but who had cervical HPV 16 or 18 infections. One of 77 women with normal cervical pathology and no cervical HPV infection was positive for HPV 16 DNA in her blood. The results indicate that HPV DNA can be detected in the blood of women with more advanced cervical carcinomas but not in the blood of women with pre-cursor cervical lesions. The results of our study indicate that the role of HPV DNA in the circulatory system appears not be of diagnostic significance and HPV DNA is only detectable in women with more advanced cervical cancers.     

临床宫颈组织标本HPV16 L1蛋白检测

 目的 检测HPV16阳性宫颈标本L1蛋白的表达,发掘宫颈损伤程度与L1蛋白表达的规律。方法 PCR法对宫颈炎、宫颈上皮内瘤变（CINⅠ~Ⅱ）、原位癌、早期浸润癌及中晚期癌标本进行型别检测,ELISA法及免疫组化法检测HPV16阳性标本中HPV L1蛋白表达。结果 54例宫颈组织中HPV16阳性为46例（85.2%）。随着宫颈损伤加重,HPV16阳性标本的抗原抗体反应逐渐减弱;免疫组化阳性反应仅出现于上皮组织内,反应强度及分布与宫颈损伤程度及癌变组织的分化状态有关。结论 HPV16 L1蛋白的表达同时受宫颈组织损伤程度及宿主细胞分化状态的影响。早期对HPV L1蛋白进行检测,可为宫颈癌的早期预防及临床诊治提供理论指导。     

A comparison of methylation levels in HPV18, HPV31 and HPV33 genomes reveals similar associations with cervical precancers

BackgroundHigh risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool.ObjectivesOur aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study.Study designPyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population.ResultsAfter adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant.ConclusionElevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections.     

Mucosal vaccination with a recombinant Salmonella typhimurium expressing human papillomavirus type 16 (HPV16) L1 virus-like particles (VLPs) or HPV16 VLPs purified from insect cells inhibits the growth of HPV16-expressing tumor cells in mice

Human papillomaviruses, mainly type 16 (HPV16), are responsible for cervical intraepithelial neoplasia, which can lead, in association with other factors, to cervical cancer. Both Salmonella recombinant vaccine strains assembling HPV16 virus-like particles (VLPs) and HPV16 VLPs purified from insect cells are able to induce HPV16 neutralizing antibodies in genital secretions of mice after nasal immunization. Anti-HPV16-specific antibodies in cervical secretions of women may prevent genital infection with HPV16, although this cannot be critically evaluated in the absence of an experimental model for genital papillomavirus infection. Induction of HPV16-specific cell-mediated immunity in the genital mucosa could improve the efficacy of a vaccine and a mucosal route of immunization might be necessary to do so. It has been shown that systemic immunization of mice with purified HPV16 VLPs confers protection against an HPV16-expressing tumor cell challenge through the induction of cytotoxic T-lymphocytes. Using the same C3 tumor model, we show that intranasal immunization of mice with purified HPV16 VLPs in a prophylactic setting also induces anti-tumor immunity. More interestingly, mucosal vaccination of mice with a Salmonella recombinant strain stably expressing HPV16 L1 VLPs also induces anti-tumor immunity in prophylactic as well as in therapeutic settings. Our data suggest that attenuated Salmonella strains expressing chimeric VLPs containing nonstructural viral proteins might be a promising candidate vaccine against cervical cancer by inducing both neutralizing antibodies and cell-mediated immunity.     

人乳头状瘤假病毒中和抗体滴度和ELISA法测定的小鼠血清抗体滴度的相关性研究

目的 探讨两种不同的报告基因纽扣珊瑚绿色荧光蛋白(Zoanthus sp.green fluorescent protein,ZsGreen)和分泌性碱性磷酸酶(secreted alkaline phosphatase,SEAP)测量的人乳头状瘤假病毒中和滴度之间的相关性,以及中和滴度和抗体滴度的相关性.方法 将密码子优化的人乳头状瘤病毒(HPV)衣壳蛋白L1、L2基因表达质粒和报告基因质粒共转染293FT细胞,48 h后收集细胞裂解上清,柱层析纯化假病毒,对假病毒滴度进行测定.采集免疫过候选HPV疫苗和Gardasil疫苗的小鼠血清,测量血清的中和滴度和抗体滴度.结果 经统计分析,这两种报告基因系统的假病毒检测的中和滴度结果高度相关(Spearman相关系数r=0.760),而且中和滴度和抗体滴度的相关度很高(Spearman相关系数r=0.577和0.741).结论 两种不同的报告基因ZsGreen和SEAP测量的HPV假病毒中和滴度之间,以及中和滴度和ELISA测定的抗体滴度之间高度相关,揭示了部分HPV疫苗预防病毒入侵的机制,为快速准确鉴定HPV-16和HPV-18候选疫苗的免疫保护效果奠定了基础.     

Accurate human papillomavirus genotyping by 454 pyrosequencing

Accurate HPV typing is essential for evaluation and monitoring of HPV vaccines, for second-line testing in cervical cancer screening, and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons, generated by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12 800-fold coverage, the NGS method allowed us to correctly identify all high-risk HPV types, in either single or multiple infections, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types.     

Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay

PCR methods enable the detection of a large variety of human papillomavirus (HPV) genotypes that infect the anogenital tract. However, PCR with consensus primers, general primers, and, to a lesser extent, broad-spectrum primers may underrepresent the true prevalence of HPV, especially the true prevalence of multiple infections. We compared the rate of HPV positivity determined by a broad-spectrum PCR with primers BSGP5+ and BSGP6+ (BS-PCR) coupled to an established bead-based multiplex HPV genotyping (MPG) assay with the rate of HPV positivity determined by a multiplex PCR with type-specific primers (TS-PCR) coupled to a newly developed MPG assay for 735 selected cervical scraping samples. While the primers used for the BS-PCR are located within the L1 region of the HPV genome, the primers used for the TS-PCR target the E7 gene. The overall rates of positivity for the 19 HPV types included in both assays were 60.9% and 72.2% by the BS-PCR and the TS-PCR, respectively, and the two assays found multiple infections in 34.8% and 58.0% of the specimens, respectively. Both HPV detection assays allowed the semiquantitative detection of HPV types and identified the same dominant HPV type in 66.6% of the multiple infections. In conclusion, the TS-PCR-MPG assay significantly increased the rate of detection of HPV DNA and the number of infections with multiple HPV types detected and demonstrated that the prevalence of low-copy-number HPV infections in the anogenital tract may be strongly underestimated by conventional HPV amplification methods, especially in cases of multiple infections. As a consequence, PCR-TS-MPG appears to be highly suited for analysis of the significance of multiple infections in the development of cervical cancer and for the study the natural history and the latency of HPV.Human papillomaviruses (HPV) are DNA viruses that infect cutaneous and mucosal epithelia. Until now, approximately 100 HPV genotypes have been fully characterized on the basis of the isolation of complete genomes (7), and there is evidence that a larger number exist (1). There are approximately 45 known mucosal HPV types; and these are further divided into three groups on the basis of their epidemiological association with cervical cancer: high-risk HPV (Hr-HPV) types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82), putative high-risk HPV (pHr-HPV) types (types 26, 53, and 66), and low-risk HPV (Lr-HPV) types (e.g., types 6, 11, 40, 42, 43, 44, and 70) (18). Hr-HPV types are causally associated with several malignant diseases, of which cervical cancer has particular significance, being the second most common cancer in women worldwide and the main cancer of women in most developing countries (18). Hr-HPV type DNA has been detected in 99.7% of cervical cancer tissue specimens (26), and persistent infection with an oncogenic HPV type, particularly HPV type 16 (HPV-16) or HPV-18, is recognized as a necessary cause of cervical cancer. HPV genotyping is of importance for the study of the natural history of infections with one or several HPV types and the role of HPV persistence in the progression of cervical lesions and for the monitoring of vaccine efficacy.Among HPV-positive women, 20 to 40% harbor in their cervices at least two types that were acquired simultaneously or successively (17). It remains controversial whether an infection with multiple types (referred to here as a multiple infection) is a risk factor for the persistence of HPV and for cervical lesions (20, 21). Moreover, it remains unknown whether women with, e.g., quadruple infections are at higher risk than women with double infections. Interest in multiple HPV infections has recently increased as prophylactic vaccines against HPV types 6, 11, 16, and 18 are expected to also provide partial protection against related HPV types by cross-neutralizing antibodies (12). Therefore, it is important to accurately type all HPV infections present in one patient. It will also be of particular interest to study the long-term impact of vaccination on the established equilibrium in the distribution of HPV types within immunized populations. Therefore, the sensitive, reliable, and unbiased profiling of the individual HPV types in patients with multiple infections is important to learn more about the natural history of HPV and to evaluate the effect of HPV vaccination.HPV typing based on PCR methods has continuously been improved over the past few years. One of the most common PCR uses the GP5+ and GP6+ primer pair, which targets conserved sequences within the L1 region of the virus genome flanking highly variable type-specific sequences (6). Use of PCR with this primer pair allows the amplification of a broad range of mucosal HPV types in a single reaction. The HPV genotype can be determined by analyzing the PCR product generated by sequence analysis, restriction fragment length polymorphism analysis, or hybridization with type-specific probes in different formats, such as the reverse line blot (RLB) assay (24) or the bead-based multiplex HPV genotyping (MPG) method (22). Recently, the performance of the PCR with primers GP5+ and GP6+ has been improved by addition of eight forward broad-spectrum primers and two backward broad-spectrum primers (primers BSGP5+ and BSGP6+ [BS-PCR]), which has led to a more homogeneous analytical sensitivity for the detection of genital HPV types (23). Consequently, the detection of HPV types 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, as well as the detection of multiple infections, was significantly improved by BSGP5+/BSGP6+ MPG. In addition, it has been shown that not only the use of degenerate primers (primers GP5+ and GP6+) (6) and/or consensus primers (primers MY09 and MY11) (2) but also the use of broad-spectrum primers (14) may lead to the underdetection of low copy numbers of HPV, particularly in cases of multiple infections (19).Recently, a highly sensitive, multiplex type-specific HPV E7 PCR system detecting a larger proportion of multiple infections than GP5+/GP6+ RLB has been described (9). However, the detection of PCR products was based on an array primer extension (APEX) assay, a time- and labor-intensive procedure with little high-throughput ability.In this report, we describe the development of an ultrasensitive and highly specific Luminex-based assay for the genotyping of products from the multiplex type-specific E7 PCR (TS-PCR). The results obtained by this novel high-throughput assay were compared to those obtained by the already established, sensitive BSGP5+/BSGP6+ MPG assay.     

含密码子优化型HPV16 L1基因重组腺病毒的构建及其免疫效果的研究

目的 构建含密码子优化型HPV16L1基因的重组腺病毒,对其经不同接种途径所诱导的系统性及黏膜免疫效果进行研究.方法 使用Admax系统包装重组腺病毒,纯化的腺病毒以不同方式免疫C57BL/6小鼠,间接ELISA及体外中和实验检测免疫小鼠血清及阴道分泌物中的特异性抗体.结果 重组腺病毒滴鼻接种可同时诱导特异性的系统性及黏膜免疫反应,重组腺病毒肌注免疫仅能诱导系统性免疫反应,而阴道黏膜接种不能有效诱导系统性及黏膜免疫反应.结论 成功构建了含密码子优化型HPV 16 L1基因的重组腺病毒,重组腺病毒肌注可诱导高滴度的血清中和抗体,滴鼻接种可同时诱导特异性的系统性及黏膜免疫反应.     

Expression of HPV L1 capsid protein in cervical specimens with HPV infection

We tried to investigate the expression rate of human papillomavirus (HPV) L1 capsid protein in uterine cervical specimens and correlate it with the grade of dysplasia, HPV genotype and age of the patients. Among uterine cervical specimens proved to have HPV by DNA genotyping test, eighty cytology-biopsy matched cases and 22 unmatched cytology specimens were selected. Immunostaining for L1 capsid protein was performed on both cervical smears and tissue sections. The L1 capsid protein was expressed mainly in the nuclei, but occasionally in the cytoplasm of cells located in the superficial layer of squamous epithelium. The immunostaining for L1 capsid protein showed positive reaction in 47 cases (46.1%) of cervical smears and in 10 cases (12.5%) of tissue sections (P = 0.001). Cytologic diagnosis revealed a higher expression rate in LSILs (25/33; 75.8%) than in HSILs and cervical cancers (8/20; 40.0% and 2/5; 40%, respectively) (P = 0.006). In LSILs, cases with low-risk type HPV showed a higher L1 capsid expression rate than those with the high-risk type HPV (88.9% vs. 70.8%). The L1 capsid expression rate decreased in the over-40-year-old age group compared to the younger age (49.2% vs. 50.8%). Cytology smears were superior to tissue sections for the detection of L1 capsid protein expression. LSILs and HPV low-risk group showed higher L1 capsid expression rate than HSILs and HPV high-risk group, which suggests that L1 capsid expression might be related to a favorable disease biology.     

Multiple HPV genotypes in cervical carcinomas: improved DNA detection and typing in archival tissues.

BACKGROUND: Human papillomaviruses (HPV) have been considered to be the necessary and central agents of cervical carcinoma. OBJECTIVE: The aim of this study was to determine the prevalence and genotypes of HPV in archival cervical carcinomas. STUDY DESIGN: The study included 152 paraffin-embedded, formaldehyde-fixed cervical carcinoma specimens. To improve the detection and typing of HPV in archival tissues, we conducted a comprehensive study in which, polymerase chain reaction (PCR)-based methods using E7 type-specific (TS) and L1 modified general primers (MY11/GP6+ and GP5+/GP6+) were employed. RESULTS: Overall HPV prevalence was 98% in the cervical carcinomas. HPV 16 was detected in 66% of the tumors, HPV 18 in 22%, HPV 31 in 13%, HPV 33 in 9%, and HPV 58 in 9%. Notably, multiple HPV types were present in 44 (28.9%) of the 152 cervical carcinomas. The most common co-infections were HPV types 16/18 (12 cases), followed by HPV types 16/31 (7 cases). Additionally, HPV 18 was more frequent in adenocarcinomas and adenosquamous carcinomas (86%) than in squamous cell carcinomas (15.8%) (P = 0.0002). CONCLUSIONS: The combination of L1 general primers and E7 type-specific primers can be of use in detecting HPV DNA in archival tissues. The present study showed a high frequency of multiple HPV infections in cervical carcinomas. Hence, relevant HPV typing information in cervical carcinoma is very important for further HPV vaccine design and application.     

人乳头瘤病毒(HPV)L2肽免疫小鼠诱导的抗体水平与保护作用的关系研究

目的 检测HPV-58 L2 11～200 AA肽在动物体内对HPV的保护效果,并分析疫苗诱导的抗体滴度或中和抗体滴度与疫苗保护作用之间的对应关系.方法 利用大肠杆菌表达HPV-58L2 11～200 AA肽,纯化目的 蛋白并与铝佐剂吸附后免疫小鼠,利用小鼠HPV-58假病毒感染模型检测不同剂量的免疫原对小鼠的保护作用.通过ELISA和假病毒中和抗体检测方法 检测免疫血清中的总抗体水平和中和抗体水平,分析具有保护作用的抗体或中和抗体滴度.结果 当蛋白免疫剂量为8μg时,能够完全保护小鼠不受HPV假病毒的感染.小鼠免疫血清中的中和抗体水平较低,利用已建立的假病毒中和试验方法 检测不到血清中的中和抗体.而利用ELISA检测血清中的总抗体水平结果 显示,免疫血清中的总抗体水平与疫苗的保护效果之间存在对应关系,当抗体滴度大于等于1:25 000时,小鼠体内检测不到荧光信号,能够保护机体不受假病毒的感染.结论 HPV-58 L2 11～200 AA肽能够保护小鼠不受假病毒的感染,L2 11～200 AA肽具有较好的疫苗发展前景,其引发的总抗体水平可以作为评价保护效果的间接指标.