兰州地区宫颈癌组织标本中HPV16感染情况的初步分析

人乳头瘤病毒的感染与宫颈癌有密切关系。通过兰州地区宫颈癌患者的HPV感染情况的分析对HPV16与宫颈癌之间的关系进行了研究。采用套式PCR方法,以HPV DNA的早期基因E6,E7和结构基因L1为扩增目的基因,对13份兰州地区宫颈癌组织进行扩增,将扩增阳性片段测序,并与HPV16标准序列进行比较,13份组织中有12份扩增到了HPV的目的基因。证实了HPV与宫颈癌有密切关系。     

HPV16+治疗性无佐剂蛋白疫苗—HPV16Z- Hsp65-E6/E7的构建、表达及纯化工艺研究

目的：研制针对我国宫颈癌高危相关的HPV16型的治疗性无佐剂蛋白疫苗。方法：应用PCR技术自我国山西宫颈癌高发现场分离到的毒株-HPV16z中获得E6/E7转化基因片段,自卡介苗菌株中克隆获得Hsp65基因片段,对E6/E7基因片段定点突变修饰,构建pET28a-Hsp65-E6/E7表达载体,在大肠杆菌BL21(DE3)中表达融合蛋白,并研究重组蛋白的纯化方案和工艺。结果：成功构建pET28a-Hsp65-E6/E7重组表达载体,E6/E7突变位点正确,融合蛋白在亲和层析柱上正确复性和初步纯化,经阴离子交换色谱纯化后蛋白纯度达到95%。结论：该研究为无佐剂治疗性重组蛋白疫苗Hsp65-E6/E7的进一步功能研究奠定了基础。     

Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah,Saudi Arabia

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.     

Secular trends of HPV genotypes in invasive cervical cancer in Cali,Colombia 1950–1999

Aim To estimate relative contribution and time trends of HPV types in cervical cancer in Cali, Colombia over a 50 years’ period.MethodsParaffin blocks of 736 cervical cancer histological confirmed cases were retrieved from the pathology laboratory at Hospital Universitario del Valle (Cali, Colombia) and HPV genotyped using SPF10-PCR/DEIA/LiPA25 (version 1) assay. Marginal effect of age and year of diagnosis in secular trends of HPV type prevalence among HPV+ cases were assessed by robust Poisson regression analysis.Results64.7% (95%CI: 59.9–69.2) of squamous cell carcinomas (SCCs) were attributed to HPV 16 and 18, 78.2% (95%CI: 74–82) to HPV 16, 18, 31, 33 and 45 and 84.8% (95%CI: 81–88.1) to HPV 16, 18, 31, 33, 45, 52 and 58 while ninety-three percent of adenocarcinomas (ADCs) were attributed to HPV 16, 18 and 45 only. The prevalence of specific HPV types did not change over the 50-year period. A significant downward trend of prevalence ratios of HPV16 (​P = 0.017) and α7 but HPV 18 (i.e., HPV 39, 45, 68, 70, ​P = 0.024) with increasing age at diagnosis was observed. In contrast, the prevalence ratio to other HPV genotypes of α9 but HPV 16 genotypes (i.e., HPV 31, 33, 35, 52, 58, 67, ​P = 0.002) increased with increasing age at diagnosis.ConclusionNo changes were observed in the relative contribution of HPV types in cervical cancer in Cali, Colombia during the 50 years. In this population, an HPV vaccine including the HPV 16, 18, 31, 33, 45, 52 and 58 genotypes may have the potential to prevent ∼85% and 93% of SCC and ADC cases respectively.     

人乳头瘤病毒16型(新疆株)E7基因的克隆与突变研究

目的：克隆分析人乳头瘤病毒16型(HPV16)新疆株的研基因;并对E7基因进行突变改造,以比较野生型与突变型HPV16E7基因的功能。方法：根据从中国新疆维吾尔族妇女宫颈癌活检组织标本中提取的DNA,进行PCR扩增获得HPV16E7基因,然后分别将其克隆到pMD18-T载体上进行DNA序列分析。根据HPV16E7基因的特点,分别设计点突变引物,用PCR的方法进行HPV16E7基因的点突变。结果：PCR检测显示扩增出HPV16(新疆株)E8基因;测序结果表明HPV16-XJ的研基因全长297bp,与德国标准株一致;利用设计突变位点的引物经PCR扩增,经序列测定后,分别得到了第70、172、271位碱基突变的HPV16E7基因;分别构建了野生型与单、双、三点突变的重组质粒pMD18-T-HPV16E7。结论：人乳头瘤病毒16型(新疆株)E7基因结构与德国标准株相同。HPV16E7基因多点突变的改造,为探索HPV16E7基因功能的变化和开展疫苗研究奠定了理论基础。     

Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines

'High-risk' human papilloma viruses (HPVs) cause cervical tumours. In order to treat these tumours therapeutic approaches must be developed that efficiently target the tumour cells. Using phage display, we selected tumour-targeting peptides from a library of constrained nonamer peptides presented multivalently on pVIII of M13. Three different consensus peptide sequences were isolated by biopanning on HPV16-transformed SiHa cells. The corresponding phage-peptides targeted and were internalized in HPV16 transformed SiHa and CaSki cells as well as in HPV18-transformed HeLa cells, but failed to bind a panel of normal or transformed cell lines. Two of the three selected peptides targeted cells only when presented on phage particles in a constrained conformation. However, all three peptides retained their targeting capacity when presented on the reporter protein enhanced green fluorescent protein (EGFP) in a monovalent form. These peptides may be useful for the design of drug or gene delivery vectors for the treatment of cervical cancer.     

我国宫颈癌疫苗市场分析及对策研究*

宫颈癌Cervical cancer是最常见的妇科恶性肿瘤,是威胁女性健康的第二大恶性肿瘤。宫颈癌疫苗是预防宫颈癌发病的有效途径。2019年以前葛兰素史克(GSK)和默沙东(MSD)垄断了全球宫颈癌疫苗药物市场。虽然国内宫颈癌疫苗起步较晚,但在国内创新政策驱动下,20种疫苗已进入临床阶段,特别是由厦门万泰联合厦门大学研发的馨可宁于2020年4月获批上市,它是我国首个自主研发、全球第三个的宫颈癌疫苗。相比较欧美等发达国家,我国在宫颈癌疫苗推广力度上还有较大差距,我国9~45岁女性的HPV疫苗接种率不足0.05%。面对HPV疫苗接种覆盖率低,提出了优化HPV疫苗审评审批流程、将HPV疫苗纳入国家免疫规划、提高女性对HPV疫苗的认识等对策建议。     

Human papillomavirus genotyping by multiplex pyrosequencing in cervical cancer patients from India

Cervical cancer is a leading cause of cancer-related deaths among women in India.Human papillomavirus (HPV) infection is the causative agent of cervical cancer; and infection with the high-risk genotypes, predominantly HPV16 and 18,is the biggest risk factor.Vaccines targeting HPV16 and 18 have been found to confer protection in large- scale clinical trials.HPV genotyping has traditionally been carried out to screen the population "at risk" using indirect methods based on polymerase chain reaction (PCR) using consensus primers combined with various DNA hybridization techniques,and often followed by the sequencing of candidate products.Recently,a high-throughput and direct method based on DNA sequencing has been described for HPV genotyping using multiplex pyrosequencing. We present a pilot study on HPV genotyping of cervical cancer and non-malignant cervical samples using multiplex pyrosequencing.Using genomic DNA from cell lines,cervical biopsies,surgical tissues or formalin-fixed,paraffin- embedded tissue samples,we could successfully resolve 6 different HPV types out of the 7 tested,with their prevalence found to be in agreement with earlier reports. We also resolved coinfections with two different HPV types in several samples. An HPV16 genotype with a specific and recurrent sequence variation was observed in 8 cancer samples and one non-malignant sample. We find this technique eminently suited for high-throughput applications,which can be easily extended to large sample cohorts to determine a robust benchmark for HPV genotypes prevalent in India.     

免疫印记法检测宫颈脱落细胞中HPV16/18 E6蛋白的意义

目的用免疫印记法检测宫颈脱落细胞中人乳头瘤病毒（HPV）16/18型E6蛋白的表达,从而为探求一种简捷、无创的诊断宫颈癌及其癌前病变的新方法提供理论依据。方法采用免疫印记法（Western blot）检测112例宫颈脱落细胞标本中HPV16/18 E6蛋白的表达,并以导流杂交检测标本中HPV DNA作为对照。结果在宫颈癌组和CINII/Ⅲ组,HPV16/18 E6蛋白的表达水平（75%、67.5%）明显高于正常对照组（5%）,P（0.05;而CINI组（31.5%）与正常对照组比较,HPV16/18 E6蛋白表达的差异无统计学意义,P=0.05。结论宫颈脱落细胞中HPV16/18 E6蛋白的过度表达与宫颈癌及CINII/Ⅲ的发生密切相关;利用免疫印记法检测脱落细胞中HPV16/18 E6蛋白对宫颈鳞癌及CINII/Ⅲ的诊断及无创性筛查具有重大意义。     

PCR based detection of HPV 16 and 18 genotypes in normal oral mucosa of tobacco users and non-users

There is increasing evidence of a causal association between human papillomavirus (HPV) and oral squamous cell carcinoma (OSCC). Several studies have shown that HPV is associated with increased risk of oral cancer independent of exposure to tobacco and alcohol. The association is valid for HPVs 16 and 18, which generally are considered high risk types, because they have been detected in oral dysplastic lesions and cancers. We determined the baseline prevalence of HPVs 16 and 18 in normal oral mucosa of individuals with and without tobacco habit. PCR was used for DNA collected by oral smears to detect HPV 16/18 DNA in normal oral mucosa of 60 healthy individuals who were assigned to two groups of 30 subjects each. One group had a tobacco habit, the other did not. The tobacco user group comprised individuals who were tobacco chewers only. Sixty-five percent of individuals were positive for HPV 16/18 DNA, but HPV 16/18 positivity was less in individuals with tobacco habit than in those without tobacco habit. No significant association was found between the presence of HPVs and gender, age or duration of chewing habit, or between groups with and without a tobacco habit. We propose that HPVs16 and 18 commonly are present in normal oral mucosa and emphasize the importance of distinguishing clinical, subclinical and latent HPV infections when investigating HPVs and OSCC.     

Surveillance of effects of HPV vaccination in Belgium

BackgroundEarly effects of HPV (human papillomavirus) vaccination are reflected by changes observable in young women attending cervical cancer screening.Subject and methodsThe SEHIB study included HPV geno-typing of ∼6000 continuous and 650 pathological cervical cell specimen as well as biopsies, collected from women in Belgium in 2010–2014. Data were linked to vaccination status.ResultsHPV vaccination offered protection among women aged <30 years against infection with HPV16 (vaccine effectiveness [VE] = 67%, 95% CI: 48–79%), HPV18 (VE = 93%, 95% CI: 52–99%), and high-risk HPV (VE = 16%, 95% CI: 2–29%). Vaccination protected also against cytological lesions. Vaccination protected against histologically confirmed lesions: significantly lower absolute risks of CIN1+ (risk difference [RD] = −1.6%, 95% CI: −2.6% to −0.7%) and CIN3+ associated with HPV16/18 (RD = −0.3%, 95% CI −0.6% to −0.1%). Vaccine effectiveness decreased with age. Protection against HPV16 and 18 infection was significant in all age groups, however no protection was observed against cytological lesions associated with these types in age-group 25–29.ConclusionThe SEHIB study demonstrates the effectiveness of HPV vaccination in Belgian young women in particular in age group 18–19. Declining effectiveness with increasing age may be explained by higher tendency of women already exposed to infection to get the vaccine.     

Association of sexually transmitted infections and human papillomavirus co-infection with abnormal cervical cytology among women in Saudi Arabia

Human papillomavirus (HPV) is a causative agent of cervical and other cancers. Sexually transmitted Infections (STIs) may play a crucial role in HPV persistence, leading to serious complications, including cervical cancer. This study investigated the association of HPV/STI co-infection in cervical samples with cervical dysplasia among women in Saudi Arabia. HPV-positive cervical samples (n = 142) were obtained from previous studies and newly collected samples (n = 209) were obtained from women aged 19–83 years. For HPV detection and genotyping, PCR and Genoflow HPV assay kits were used. STIs were detected using a Genoflow STD array kit. Of 351 samples, 94 (27%) were positive for STIs. Among HPV-positive samples, 36 (25%) were positive for STIs; the most common pathogens were Ureaplasma urealyticum/Ureaplasma parvu (13%) and Mycoplasma hominis (6%). A global significant correlation was detected between HPV and STIs with progression of abnormal cervical cytology (χ² = 176, P < 0.0001). Associations between cervical cytology diagnosis and HPV status, STI types (opportunistic and pathogenic), and the presence of Ureaplasma spp., and Mycoplasma hominis were significant (P < 0.05). Our results suggest that additional study in a larger population is warranted to determine the association between HPV/STI co-infection and cervical neoplasia in Saudi women.     

子宫颈癌预防用人乳头瘤病毒疫苗及其展拓

人乳头瘤病毒(HPV)是人子宫颈癌病原体,这一发现奠定了子宫颈癌预防用人乳头瘤病毒疫苗得以问世的科学基础。临床实践证明,默克(Merck)公司的Gardasil和葛兰素史克(GSK)公司的Cervarix这两种疫苗可预防由HPV16和HPV18引起的子宫颈癌,有效率几乎达100%。Gardasil和Cervarix的成功激动了围绕Gardasil和Cervar-ix的展拓研究。     

Intratype variation in 12 human papillomavirus types: a worldwide perspective.

In this study, we have examined intratype human papillomavirus (HPV) sequence variation in a worldwide collection of cervical specimens. Twelve different HPV types including HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-58, HPV-59, HPV-68 (ME180), MM9/PAP238A (recently designated HPV-73), and a novel partial genomic HPV sequence designated MM4/Wl3B were analyzed in this study. Cervical specimens were collected as part of epidemiological investigations conducted in New Mexico and an international study of invasive cervical cancer (IBSCC). Specimens from several countries including Argentina, Brazil, Bolivia, Benin, Cuba, Colombia, Chile, Germany, Mali, Panama, Paraguay, Spain, Algeria, Uganda, Guinea, Tanzania, Indonesia, Philippines, Thailand, and the United States were evaluated. Specimen DNAs were subjected to amplification with the MY09/11 L1 consensus PCR system. The PCR products were cloned, and an approximately 410-bp region in the L1 open reading frame was sequenced from 146 specimens (approximately 60,000 bp). Within a single HPV type, nucleotide diversity varied between 0.2 and 2.9% (i.e., between any pair of variants) and the majority of nucleotide changes were synonymous (amino acid conserving). These data provide information pertinent to HPV diagnostic probe development and are potentially relevant to future rational vaccine strategies. Similarly, amino acid diversity varied between 0 and 5.1%. Some of these amino acid changes may represent markers of intertype evolutionary relationships. Presuming that HPVs have evolved under the same constraints as their corresponding hosts, the limited genetic diversity observed for all HPVs studied to date may reflect an evolutionary bottleneck occurring in both virus and host populations.     

Genetic characteristics of human papillomavirus type 16, 18, 52 and 58 in southern China

Persistent infections of high-risk human papillomaviruses (HPVs) are the leading cause of cervical cancers. We collected cervical exfoliated cell samples from females in Changsha city, Hunan Province and obtained 338 viral genomes of four major HPV types, including HPV 16 (n = 82), 18 (n = 35), 52 (n = 121) and 58 (n = 100). The lineage/sublineage distribution of the four HPVs confirmed previous epidemiological reports, with the predominant prevailing sublineage as A4 (50%), A1 (37%) and A3 (13%) for HPV16, A1 (83%) for HPV18, B2 (86%) for HPV52 and A1 (65%), A3 (19%) and A2 (12%) for HPV58. We also identified two potentially novel HPV18 sublineages, i.e. A6 and A7. Virus mutation analysis further revealed the presence of HPV16 and HPV58 sublineages associated with potentially high oncogenicity. These findings expanded our knowledge of the HPV genetic diversity in China, providing valuable evidence to facilitate HPV DNA screening, vaccine effectiveness evaluation and control strategy development.     

利用重组HPV16L1抗原检测宫颈癌抗L1或VLP抗体的对比

为了评价重组大肠杆菌表达的HPV16L1蛋白和重组腺病毒表达的HPV16L1 VLP两种抗原在检测宫颈癌抗 16L1或VLP抗体及在宫颈癌血清学诊断意义上的差别 ,应用PCR技术从宫颈癌组织的DNA中扩增出全长15 35bp的HPV16L1基因片段 ,克隆至 pUC18 T载体中 ,进行DNA测序鉴定。然后 ,将HPV16L1基因克隆至pGEX 2T表达载体中 ,并诱导表达HPV16L1融合蛋白 ,分子量为 83kD ,能被HPV16L1单克隆抗体所识别。经GST柱层析法纯化后 ,与重组腺病毒表达的HPV16L1 VLP分别经酶联免疫吸附 (ELISA)法检测 12份宫颈癌患者和 35份献血员血清。 12例宫颈癌血清标本中 ,抗HPV16L1蛋白的抗体阳性率为 7例 (占 5 8.3% ) ;抗HPV16L1 VLP的抗体阳性率为 8例 (占 6 6 .7% )。经大肠杆菌表达的重组抗原HPV16L1检测为HPV16抗体IgG( )的 7份患者血清 ,利用HPV16L1 VLP试剂盒检测均阳性 ;经大肠杆菌表达的重组抗原检测为HPV16抗体IgG( )的 5份患者血清 ,利用HPV16L1 VLP试剂盒检测有 1份阳性。两者对HPV16抗体的阳性检出率并无显著差异 (P >0 .0 5 )。本实验结果说明HPV16与宫颈癌高度相关 ,利用大肠杆菌表达的重组抗原HPV16L1和HPV16L1 VLP重组抗原检测抗体的敏感性并不受影响。利用重组抗原HPV16L1对宫颈癌的抗体进行定性、定量分析有助于该疾病     

重叠PCR合成HPV16E6、E7基因并构建植物表达双元载体

HPV16型为主的多种HPV病毒可诱发机体发生宫颈癌等疾病,以重叠PCR法人工合成HPV16 E6、E7致癌基因的融合基因,以之为目的基因构建了无选择标记基因(Marker-Free)双元载体,期望转化番茄开发新型宫颈癌治疗性疫苗-转基因植物口服疫苗.通过生物信息学分析HPV16 E6、E7基因,设计并合成密码子优化的靶基因E6-E7融合基因;并在目的基因的上游引入分子佐荆LTB基因,与Kozak序列等表达元件相偶联,以提高目的基因在植物表达系统的表达水平、增强其诱导黏膜免疫的免疫原性.目前已构建pX6-LTB-E7和pX6-LTB-E7-E6两个番茄转化双元载体.采用番茄Marker-Free系统转化和表达HPV16 E6、E7目的基因可以在转化后代中剔除标记基因,从而消除由标记基因可能引起的转基因植物口服疫苗的安全性问题,为HPV转基因植物口服疫苗应用奠定基础.     

A comprehensive review on host genetic susceptibility to human papillomavirus infection and progression to cervical cancer

Cervical cancer is the second most common cancer in women worldwide. This is caused by oncogenic types of human papillomavirus (HPV) infection. Although large numbers of young sexually active women get HPV-infected, only a small fraction develop cervical cancer. This points to different co-factors for regression of HPV infection or progression to cervical cancer. Host genetic factors play an important role in the outcome of such complex or multifactor diseases such as cervical cancer and are also known to regulate the rate of disease progression. The aim of this review is to compile the advances in the field of host genetics of cervical cancer. MEDLINE database was searched using the terms, 'HPV', 'cervical', 'CIN', 'polymorphism(s)', 'cervical'+ *the name of the gene* and 'HPV'+ *the name of the gene*. This review focuses on the major host genes reported to affect the progression to cervical cancer in HPV infected individuals.     

A protective and broadly cross-neutralizing epitope of human papillomavirus L2

We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.     

Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.