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。     

人乳头瘤病毒检测方法研究进展

人乳头瘤病毒（Human papilloma viruses,HPV）是少数几种明确与人类癌症相关的病毒之一,其持续感染是导致宫颈癌发生的重要危险因素。HPV的检测对宫颈癌的筛查及治疗宫颈病变药物的有效性评价有重要作用,因此国内外对HPV的检测方法不断革新。纵观近年来HPV检测方法,主要依赖于分子生物技术检测HPV的DNA、RNA、癌蛋白等,但各种检测方法的原理、适用范围及临床意义均不相同。本文将就目前常用的HPV检测技术及试剂盒进行简要介绍,并对各方法的优缺点、灵敏度及特异性进行分析,以期为临床选择合适的检测方法提供参考。     

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 μg/L, with 100 μg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.     

553 例女性宫颈高危型HPV 感染与宫颈病变的相关性研究

目的:研究女性生殖道高危型人乳头瘤病毒(high risk human papilloma virus,hrHPV)感染现状及子宫颈上皮内瘤样病变(cervical intraepithelial neoplasia,CI N)的现患率,并分析该人群年龄与HPV感染及宫颈癌前病变的相关性以及hrHPV病毒载量与宫颈病变之间剂量-效应关系。方法:对553例30岁-78岁有性生活史的女性进行以人群为基础的横断面调查。对所有接受筛查的妇女均行宫颈HPV检测及电子阴道镜下病理活检,并以病理结果作为诊断子宫颈病变的金标准。资料采用EXCEL整理,利用x2检验和非条件Logistic回归分析危险因素和CI N的关系。结果:553例受检对象总体阳性检出率为18.0%。hrHPV感染情况在不同年龄段分布有显著差异,hrHPV病毒载量越高宫颈病变的程度越重。结论:不同年龄段hrHPV感染情况不同,hrHPV病毒载量与宫颈病变之间存在剂量-效应关系。     

膦甲酸钠对宫颈高危HPV持续感染患者病毒载量的影响

目的 探讨抗病毒药物膦甲酸钠对宫颈高危型人乳头瘤病毒（HPV）持续感染患者的病毒载量及HPV转阴率的影响,指导临床实践。方法 选择2015年1月至2015年6月112例宫颈高危型HPV持续感染患者为研究对象,在充分知情同意的前提下,患者自愿选择进入治疗组或观察组,治疗组患者随机分成膦甲酸钠组和干扰素组。最终分组情况为：观察组43例,膦甲酸钠组34例,干扰素组35例。观察组患者不采取任何干预措施仅随访观察,治疗组患者分别予以膦甲酸钠静脉滴注3.0 g/次,1次/d,连用14 d;重组人干扰素a2b肌肉注射300万U/次,隔日1次,总共10次。于治疗后第1、3、6个月采用二代杂交捕获术（HC-2）对患者进行HPV DNA半定量分析,比较各组患者病毒载量的变化及HPV转阴率。结果 第1、3、6个月治疗组患者HPV病毒载量较治疗前明显下降,HPV转阴率显著高于观察组;膦甲酸钠组患者HPV病毒载量下降幅度显著大于干扰素组;观察组患者各时间点HPV病毒载量无明显变化。治疗后第1、3、6个月,膦甲酸钠组患者有效率分别为47.06%、61.76%、79.41%,均显著高于观察组及干扰素组（P<0.01）。在随访过程中,观察组患者未发现HPV转阴病例,治疗组转阴率为30.04%,差异有统计学意义（P<0.05）;膦甲酸钠组患者转阴率为35.29%,干扰素组为25.71%,两组差异无统计学意义（χ2=0.75,P=0.27）。结论 宫颈高危HPV持续感染患者自然清除率低,建议采取积极干预措施,膦甲酸钠能降低HPV病毒载量,促进HPV转阴,对持续性高危HPV感染患者有一定治疗意义。     

高级别上皮内瘤变和不同型别人乳头瘤病毒感染患者阴道微生态特征分析

探讨高级别上皮内瘤变（high level squamous intraepithelial lesion,HSIL）患者阴道微生态的特征及不同型别人乳头瘤病毒（human papilloma virus,HPV）感染对阴道微生态的影响。

收集我院健康女性（对照组,n=54）和HSIL患者（HSIL组,n=54）的阴道分泌物样本,提取其DNA,通过HPV试剂盒检测HPV型别,并使用16S rRNA基因高通量测序分析阴道菌群的组成结构及特征。

HSIL组女性HPV阳性率为88.9%,对照组女性HPV阳性率仅有11.1 %。与对照组相比,HSIL组女性阴道乳杆菌属相对丰度降低,加德纳菌属、普雷沃菌属、双歧杆菌属相对丰度增多,但差异均无统计学意义（均P＞0.05）。与HPV Others组相比,HPV CR组女性阴道阿托波菌属相对丰度显著降低（t=2.067,P=0.043）;加德纳菌属、链球菌属、肠球菌属相对丰度降低,双歧杆菌属相对丰度升高,但差异均无统计学意义（均P＞0.05）。

HSIL患者HPV感染率升高,不同型别HPV感染会对阴道微生态产生影响,提示对阴道微生态进行干预可能对宫颈病变产生调节作用。

     

人乳头瘤病毒16型E5与IL-12联合基因疫苗的免疫活性

为了研制人乳头瘤病毒16型(HPV16)防治性疫苗,分析了HPV16 E5与IL-12联合基因疫苗的免疫活性。将构建的pcDNA3.1(+)/E5与pcDNA3.1(+)/IL-12联合免疫BALB/c小鼠,以ELISA测定小鼠血清中抗HPV16 E5 IgG水平、小鼠脾细胞培养上清中IFN-γ和IL-4含量;MTT法检测脾淋巴细胞增殖反应。结果显示末次免疫后,联合基因疫苗组和单基因疫苗组血清IgG A450值分别明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);且联合基因疫苗组显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组的IFN-γ和IL-4含量分别均明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组IFN-γ和IL-4含量(P<0.01),且联合基因疫苗组含量显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组脾淋巴细胞刺激指数(SI)分别显著高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);联合基因疫苗组与单基因疫苗组比较,SI差异无统计学意义(P>0.05)。结果表明HPV16 E5单基因疫苗以及与IL-12联合基因疫苗均能刺激机体产生较强的免疫应答,且联合基因疫苗优于单基因疫苗。     

Immune parameters of mice bearing human head and neck cancer

A xenogeneic human head and neck squamous cell carcinoma (HNSCC) model in immunocompetent mice was evaluated for its requirement of cyclosporine for progressive tumor growth. Tumor growth and T cell functions were assessed in mice receiving cyclosporine treatment for various lengths of time. Tumor cells were injected s.c. on day 1 and cyclosporine was injected i.p. daily on days 1, 1–7, 1–14, 1–21, or for the entire 28 days of tumor growth. All mice developed tumors. These tumors were confirmed to be squamous carcinomas of human origin histologically and by positive staining for human MHC class I antigen expression. Tumors were largest in mice that received cyclosporine for days 1–21 or days 1–28. Increased tumor size was associated with increased serum levels of tumor-reactive antibodies, an increased intratumoral frequency of CD4⁺ and CD8⁺ cells, but a diminished production of interleukin-2 (IL-2) by the tumor infiltrate. Also correlating with increasing tumor size was splenomegaly, a decline in the frequency, but not the absolute levels, of splenic CD4⁺ and CD8⁺ cells, and a diminished capacity to proliferate in response to concanavalin A and to be stimulated to secrete IL-2. The HNSCC tumors contributed to the immune decline since T cell functions were more depressed in the tumor bearers than in control mice receiving only cyclosporine treatment. These results demonstrate that human HNSCC tumor xenografts can grow in mice even with limited cyclosporine treatment, and that the survival of these xenografts may, in part, be due to a tumor-induced decline in select T cell functions.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health     

Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts

Summary Human corneal fibroblasts were infected with a retroviral delivery vector containing the E6 and E7 genes from human Papilloma virus type 16 in order to produce cell lines that have an expanded lifespan in culture. Morphologically, some of the trasfected corneal fibroblast lines appeared to have the normal spindle-shape morphology of diploid fibroblasts, whereas other lines appeared to have a more elongated morphology. All the cell lines were anchorage-dependent. Cells that had a normal morphology grew at a rate similar to normal diploid human corneal fibroblasts and had a population doubling time of 48 h. All E6/E7 expressing cell lines, regardless of morphology, produce types I, III, and V collagen, at levels similar to those observed in the parent corneal diploid fibroblast. These corneal fibroblast lines will be a usefulin vitro system to study collagen expression and fibril formation, as well as normal stroma development. These results also demonstrate that the use of E6/E7 genes to expand a cell’s lifespan can be a powerful tool because it does not appear to alter either the growth rate of the cell or collagen expression.     

Human papilloma virus transformed CaSki cells constitutively express high levels of functional SerpinB2

Many malignant tissues, including human papilloma virus (HPV)-associated cancers, express SerpinB2, also known as plasminogen activator inhibitor type-2 (PAI-2). Whether SerpinB2 is expressed by the HPV-transformed cancer cells, and if so, whether SerpinB2 is mutated or behaves aberrantly remains unclear. Here we show that HPV-transformed CaSki cells express high levels of constitutive wild-type SerpinB2, with cellular distribution, glycosylation, secretion, cleavage, induction and urokinase binding similar to that reported for primary cells. Neutralization of secreted SerpinB2 failed to affect CaSki cell migration or growth. Lentivirus-based over-expression of SerpinB2 also had no effect on growth, and we were unable to confirm a role for SerpinB2 in binding or regulating expression of the retinoblastoma protein. CaSki cells thus emerge as a useful tool for studying SerpinB2, with the physiological function of SerpinB2 expression by tumor cells remaining controversial. Using CaSki cells as a source of endogenous SerpinB2, we confirmed that SerpinB2 efficiently binds the proteasomal subunit member β1.     

人乳头瘤病毒的致瘤性及机制

高危型人乳头瘤病毒(HPV)可能引发多种癌症,公认的如宫颈癌和宫颈上皮内瘤变.近年来的研究表明,HPV还与头颈部鳞癌、食管癌及乳房癌等的发生密切相关.HPV引起头颈部鳞癌的机制在某种程度上与宫颈癌相似,但又有所不同.因此,阐明HPV的致癌机制对于HPV相关肿瘤的治疗具有重要意义.     

热激蛋白gp96免疫学功能及在主动免疫治疗肿瘤和传染病中的应用

热激蛋白gp96可特异性结合来源于肿瘤和病毒的抗原肽,与抗原呈递细胞表面CD91等受体作用进入胞内,并在内质网中将结合的抗原通过抗原呈递链呈递给MHCI类分子,激活特异性T细胞。同时,与细胞表面Toll样受体(TLR)TLR2、TLR4等相互作用,激活天然免疫。近期研究发现调节性T细胞(Treg)对gp96免疫功能有显著抑制作用,随着对影响gp96免疫功能的免疫抑制机制的深入了解,以及利用汉逊酵母表达有免疫活性的全长gp96蛋白体系的建立,gp96将在治疗肿瘤及传染性疾病中发挥更大的作用。     

Measures of economic advantage associated with HPV-positive head and neck cancers among non-Hispanic black and white males identified through the National Cancer Database

BackgroundNational trends show dramatic increases in the incidence of HPV-related head and neck squamous cell carcinomas (HNSCCs) among black and white males. Using cases identified through the National Cancer Data Base, we assessed factors associated with HPV 16- or 16/18 positive HNSCCs among non-Hispanic black and white males diagnosed in the U.S. between 2009 and 2013.MethodsThis sample included 21,524 HNSCCs with known HPV status. Adjusted relative risks (RRs) and 95% confidence intervals (CIs) were estimated using log-binomial regression.ResultsCompared to those with HPV-negative tumors, male patients diagnosed with HPV-positive HNSCCs were non-Hispanic white, younger at diagnosis, lived in zip-code areas with higher median household income and higher educational attainment, had private health insurance and no reported comorbidities at diagnosis. Although the risk of HPV-positive HNSCCs increased with measures of higher area-level socioeconomic status, the effect was stronger for non-Hispanic black males (RR_Adjusted = 1.76, 95% CI 1.49–2.09) than for whites (RR_Adjusted = 1.12, 95% CI 1.08–1.16). The peak age for diagnosis of HPV-positive HNSCCs occurred in those diagnosed at 45–49 years (RR_Adjusted = 1.57, 95% CI 1.42–1.73). Oropharyngeal tumors were strongly associated with HPV-positivity (RR_Adjusted = 4.32, 95% CI 4.03–4.63). In the analysis restricted to oropharyngeal anatomic sites, similar patterns persisted.ConclusionIn our analysis, measures of economic advantage were associated with an increased risk of HPV-positive HNSCCs. In order to develop effective interventions, greater understanding of the risk factors for HPV-positive HNSCCs is needed among both high-risk males and their healthcare providers.     

常州地区妇女HPV 感染状况调查

目的:研究常州地区普通妇女人群中HPV感染状况,为宫颈癌的预防及治疗提供理论依据。方法:采用PCR与基因芯片技术,对常州地区参加妇科体检的744名妇女进行HPV分型检查,并对不同分型感染情况进行统计学分析。结果:744名妇女中共检出HPV阳性者157例,感染率为21.10%。在高危型HPV感染妇女中,共计83.64%的妇女感染了以下六种亚型,依次是16型46例,58型28例,33型24例,18型18例,31型11例以及52型11例。结论:鉴于高危型HPV与宫颈癌发生的密切关系,对普通妇女人群展开HPV检测具有预防与治疗意义。     

职业献血员中人类T淋巴细胞白血病病毒感染状况的分析

了解陕西,甘肃,青海,宁夏四省区职业献血员中人类T淋巴细胞白血病病毒（HTLV）的感染状况,以便为在职业献血员中开展HTLV普检提供一定的参考依据。采用双抗原夹心ELISA法检测了四省区10个单采浆站的7416份血浆样本。结果 HTLV（1＋2）抗体阳性7例,总阳性率0．09％,其中男性4例,感染率0．09％,女性3例,感染率0．10％;18－25年龄组2例,感染率0．09％,25－35年龄组4例,感染率0．10％,35－45年龄组1例,感染率0．08％。经过分析比较发现,HTLV感染率在职业献血员中与所报道的正常人群的感染率无显著差异,同时发现,在职业献血员中,HTLV感染的阳性率无性别差异,无年龄差异。     

Potential antitumor applications of a monoclonal antibody specifically targeting human papilloma virus 16 E7 49-57 peptide

Our study aims to evaluate whether the approach of TCRm mAb has therapeutic potential against HPV-induced tumors. In the present study, we generated a murine IgG2a mAb 6C10 specifically recognizing HPV-16-E7(49-57) epitope (RAHYNIVTF) in the polypeptides and in complex with a MHC class I molecule. Analysis of the primary structure shows that the 6C10 Ab displays a novel sequence in the CDR of the heavy chain, compared to the sequences in the Kabat database, which suggests the Ab has completed its affinity maturation. The 6C10 Ab can specifically recognize E7 and Trx-E7(30-67) protein in ELISA, and can also specifically bind to T2 cell carrying HPV-16-E7(49-57) peptide. In the TC-1 cell tumor-bearing mouse model, 6C10 exhibits tumor suppression activity when compared to the isotype control Ab. 6C10 Ab has showed tumor-inhibition potency in a mouse model and this Ab may have the prospect of cancer therapy.     

Identification of CTL epitopes in hepatitis C virus by a genome-wide computational scanning and a rational design of peptide vaccine

Developing a peptide-based vaccine for the highly variable hepatitis C virus (HCV) remains a challenging task. Variant viruses not only escape antigen presentation but also persist in a patient as quasi-species. Such variants are often antagonistic to the responding T cell repertoire. To overcome these problems, we herein propose a cocktail vaccine consisting of a few epitope peptides, which make it possible to outpace the emergence of variant viruses. To design such a vaccine, we developed a way to identify HLA-A*2402-binding peptides efficiently by means of the computational scanning of the whole genome of the pathogen. Most of the predicted peptides exhibited strong binding to the HLA-A*2402 molecule, while also inducing CD8 T cell responses from the patients’ peripheral blood mononuclear cells (PBMCs). Peptide-induced T cells were capable of lysing HCV-expressing HepG2 cells which process antigens endogenously. The amount of HCV core antigen in the patients’ livers suggested that the lytic activity of the peptide-induced T cells was clearly in a range suitable for therapeutic use. If T cells were activated under optimal conditions by high density peptides, then they tended to be relatively tolerant of single amino acid variations for cytolysis. Finally, an analysis of the viral population isolated in Japan suggested no obvious changes due to immune evasion in the viral genome even in a host population highly biased toward HLA-A*2402. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Proteomic analysis of human papillomavirus‐related oral squamous cell carcinoma: Identification of thioredoxin and epidermal‐fatty acid binding protein as upregulated protein markers in microdissected tumor tissue

Human papillomavirus (HPV) infection has been identified as an etiologic agent for a subset of oral squamous cell carcinoma (OSCC) with increasing incidence. HPV DNA‐positivity may confer better prognosis but the related oncogenic mechanisms are unknown. For the identification of HPV relevant proteins, we analyzed microdissected cells from HPV DNA‐positive (n = 17) and HPV DNA‐negative (n = 7) OSCC tissue samples. We identified 18 proteins from tumor tissues by peptide fingerprint mapping and SELDI MS that were separated using 2‐DE. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified thioredoxin (TRX) and epidermal‐fatty acid binding protein as upregulated in HPV related tumor tissue. This study, investigating for the first time proteomic changes in microdissected HPV infected tumor tissue, provides an indication on the oncogenic potential of viruses.