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。     

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-末端保守氨基酸序列的免疫生物学性质的探讨

目的探讨HPV L1 C-末端保守氨基酸序列的免疫生物学性质。方法以合成HPV L1保守序列短肽、点突变该序列中酰胺化酶作用位点后的短肽以及15个氨基酸残基长的该序列交错合成的4个小肽免疫动物。采用T细胞增殖实验确定各肽是否含有T细胞表位；以ELISA方法确定各肽有无B细胞表位及有效免疫位点。结果不同肽免疫鼠T细胞增殖实验差异无统计学意义（P〉0．05），两短肽免疫兔血清中IgG抗体滴度显著强于4个小肽，但兔和鼠分泌物中均未测出抗体IgG。两短肽的免疫血清对相应短肽的反应明显强于交叉反应的强度。原序列短肽的免疫血清对来自于该短肽的N-末端序列短肽的反应略强，而点突变酰胺化酶作用位点后短肽的免疫血清对C-末端序列的短肽反应略强。结论HPV L1保守序列短肽和点突变酰胺化酶作用位点后短肽在诱导体液免疫的能力方面相似，但在交叉免疫反应性及有效免疫位点方面存在差异；两短肽的免疫原性显著强于4个小肽。     

HPV L1保守序列多肽抗血清降解HPV6感染能力的测试

目的：明确对不同类型人乳头瘤病毒主要衣壳蛋白（HPV L1）具有交叉反应性的HPV L1 C-末端保守序列抗体是否具有降解HPV6感染能力的作用。方法：收集尖锐湿疣标本,从收集的标本中找出HPV6型感染病例,并提取病毒。将多肽抗血清按不同比例稀释,与得到的病毒共同培养并中和,接触单层培养的人永生化角质形成细胞,PCR检验经HPV6病毒接触后人永生化角质形成细胞中HPV6 DNA含量,ELISA检验此细胞中HPV6 L1蛋白存在与否。结果：经不同稀释浓度抗血清中和的HPV6病毒感染的人永生化角质形成细胞,其DNA提取物的PCR产物经电泳,在凝胶280 bp处,HPV6特异性区间呈现不同强度的阳性条带,阳性条带的强度随抗血清浓度的升高逐渐降低。ELISA对经抗血清中和病毒接触的人永生化角质形成细胞提取蛋白进行测试,HPV L1蛋白表达量显示出与上述PCR检测结果同样的梯度变化趋势,变化差异具有统计学意义。结论：初步证明,HPV6 L1保守序列多肽抗血清可部分降解该病毒的感染,该抗血清具有针对更多型HPV进行中和研究的价值。     

人乳头瘤病毒L2氨基端蛋白的同源性及其交叉反应特性研究

目的 研究人乳头瘤病毒(HPV)次要衣壳蛋白(L2)在临床常见HPV感染型别中的同源性及其交叉反应特性.方法 采用生物信息学方法对临床常见HPV感染型别中的L2氨基酸序列进行比对,发现其氨基端1～200序列具有高度同源性.采用PCR法从宫颈癌患者组织DNA中扩增HPV16 L2(1～200)肽段的碱基序列,将其克隆至原核表达载体PGEX-4T-1得到重组质粒PGEX-4T-1-HPV16 L2(1～200).将测序鉴定正确的重组质粒转入E.coli BL21(DF3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE和Western blot鉴定;进一步通过Western blot检测HPV16L2(1～200)融合蛋白与HPV6、11、16和18型DNA检测阳性临床患者血清的特异性结合能力;以镍螯合亲和层析柱(Ni-NTA Agarose)纯化的HPV16 L2(1～200)融合蛋白作为包被抗原,采用间接ELISA法对98例尖锐湿疣患者、135例宫颈癌患者及96例健康对照者血清进行特异性血清IgG抗体检测.结果 在HPV6、11、18、35等14个临床常见型别中,与HPV16 L2的1～200间的氨基酸序列比较,同源性达到52.7%～74.3%;成功构建了含有HPV16 L2(1～200)的重组质粒PGEX-4T-1-HPV16 L2(1～200),含有HPV16 L2(1～200)的融合蛋白在原核表达体系中呈高效表达,表达量占总蛋白的22.6%;表达产物的相对分子质量(Mr)约49×103,与预期Mr相符;以HPV6、11、16和18 DNA阳性患者血清为一抗进行Western blot分析,结果显示,在Mr约49×103处出现特异性条带.ELISA结果显示,尖锐湿疣组、宫颈癌组患者血清及健康对照者血清抗体均值分别为0.753±0.262、0.756±0.274和0.178±0.157,阳性率分别为89.8%、88.9%和9.4%.三组间血清抗体均值及阳性率比较差异均具有统计学意义(P均＜0.001),而尖锐湿疣组和宫颈癌组间的血清抗体均值比较差异无统计学意义(P＞0.05).结论 HPV L2 N端1～200氨基酸序列具有高度同源性,并在HPV6、11、16和18型别间具有交叉反应.     

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.     

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.     

人乳头状瘤病毒16型不同基因疫苗联合免疫的实验研究

目的 探索联合免疫策略在预防和治疗人乳头状瘤病毒16型（HPV16）相关肿瘤中的作用。方法 在C57BL／6动物模型中，观察了表达HPV16基因的融合蛋白L2E7疫苗和重组痘苗病毒mE67疫苗的不同联合免疫方式在预防和治疗HPV16相关肿瘤中的作用。用酶联免疫斑点（ELISPOT）和细胞毒T淋巴细胞（CTL）反应评价它们在诱发机体产生细胞免疫应答中的作用。结果 我们发现以HPV16 L2E7融合蛋白＋佐剂（CpG）初免，用重组痘苗病毒rVVmE67加强免疫的联合免疫方式在C57BL／6小鼠实验中可以有效预防和治疗HPV16相关肿瘤的攻击。ELISPOT可以检测到高水平的E749-57肽特异性，分泌IFN-γ的效应T细胞。CTL检测同样反应出这种联合免疫方式所诱发的CTL细胞可以有效识别并杀伤含HPV16E6／E7的靶细胞。结论 以HPV16L2E7融合蛋白＋CpG初免，用重组痘苗病毒rVVmE67加强的联合免疫策略可以有效防治HPV16相关肿瘤，为进一步研究提供了科学基础。     

人乳头瘤病毒16型E2蛋白表达、纯化及抗血清制备

目的 表达人乳头瘤病毒16型(HPV16) E2蛋白,并制备小鼠抗HPV16 E2血清.方法 采用PCR技术扩增HPV16E2基因,构建入pET21b载体,重组表达载体pET21 b-HPV16E2经鉴定后转化大肠埃希菌BL21(DE3),诱导表达并鉴定表达产物.经纯化、变性和复性方法,制备可溶性HPV16 E2蛋白.免疫BALB/c小鼠制备抗血清,检测小鼠IFN-γ、CD4+T细胞、CD8+T细胞、CD4/CD8比值和抗血清滴度变化.结果 酶切和测序结果表明pET21b-HPV16 E2构建成功.表达蛋白相对分子质量(Mr)为42 000,Western blot法证明具有较高特异性.小鼠抗血清效价升高,CD4+T细胞数量和CD4/CD8比值升高,小鼠IFN-γ无升高.结论 成功制备可溶性HPV16 E2蛋白和小鼠抗HPV16 E2高效价的抗血清.     

HPV16E7E6抗原表位嵌合体DNA抗肿瘤疫苗的研究

目的:探讨HPV16 E7、E6抗原表位与HSP70 N端重组DNA疫苗抗肿瘤活性.方法:以重叠PCR将HPV16 E7基因N端60个氨基酸的编码序列与E6基因的10个(48～57)氨基酸的编码序列及HSP70 N端序列进行融合.以pcDNA3.0为载体,构建真核表达载体pcD-E76HSP.重组质粒免疫C57BL/6小鼠后,通过淋巴细胞增殖实验和细胞毒性杀伤实验研究该疫苗激发的细胞免疫反应及反应强度;观察该疫苗对C57BL/6小鼠TC-1肿瘤细胞移植瘤的治疗效果.结果:重组DNA疫苗免疫C57BL/6小鼠后,小鼠脾淋巴细胞体外增殖明显,并可诱导产生针对TC-1肿瘤细胞的特异性CTL反应;体内抑瘤试验显示该疫苗对HPV16病毒转化的TC-1细胞小鼠移植瘤的生长有抑制作用.结论:该疫苗能激发特异性细胞免疫反应,显著抑制HPV16转化的TC-1肿瘤细胞生长.     

Preliminary research of induction of the multiple HPV antibody by HPV L1 type conserved sequence aimed at human papillomavirus major protein

To investigate whether a conserved sequence of the human papillomavirus(HPV) L1 protein consisted of 12 amino acid residue can induce the antibody aimed at multiple HPV types, we screened a conserved sequence of the HPV L1 protein by forecasting B cell epitope and comparing multiple sequences. The peptide was synthesized, mixed with Freund adjuvant, and used to immunize rabbits, and those in the control group were only immunized with Freund adjuvant. Then the antibody titer was identified by indirect enzyme-linked immunosorbent assay (ELISA). And immunocytochemistry, immunofluorescence, western blot and immunohistochemistry were used to detect whether the antibody could react with cervical cancer cell lines and cervical tissue that had been identified with HPV infections. We found that the antibody titer was greater than 1:25600. Moreover, we confirmed that the antibody could react with cervical cancer cell lines and cervical tissue with HPV infections. The results showed that the peptide could induce antibody aimed at multiple HPV types. Our findings have great significance in further research of the broad spectrum HPV, HPV L1 diagnosis kits.     

Type-specific and cross-reactive epitopes in human papillomavirus type 16 capsid proteins.

Genital human papillomavirus (HPV) 16 infection is frequently associated with cancer of the uterine cervix, as well as with precancerous lesions. In order to generate serologic reagents which might be useful in the diagnosis of HPV 16 infection, rabbit polyclonal and mouse monoclonal antisera were raised to carboxy terminal peptides from the HPV 16 L1 and L2 open reading frames (ORFs). Anti-L1 and -L2 peptide sera recognized HPV 16 L1 and L2 fusion proteins in Western blots and by immunoprecipitation. In Western blot analysis of L1 proteins from different HPV types, antisera to the L1 peptide reacted only with HPV 16, thus identifying an HPV 16 type-specific linear epitope. Anti-L2 peptide sera reacted with L2 fusion proteins from HPVs 6 and 16, but not from BPV, thus identifying a partially cross-reactive epitope in the HPV 16 L2. Computer analysis of carboxy terminal amino acid sequences of the L1 and L2 ORFs of multiple HPV types supported the Western blot findings. Despite the HPV 16 type specificity found in Western blots, anti-L1 peptide sera identified nuclear antigen by immunocytochemistry in cervical biopsies infected with HPV 16, as well as other genital HPV types. Anti-L2 peptide sera failed to recognize antigen in infected tissue.     

Levels of immunoglobulin G antibodies against defined epitopes of the L1 and L2 capsid proteins of human papillomavirus type 6 are elevated in men with a history of condylomata acuminata.

Sera from 159 men attending the sexually transmitted disease clinic at Karolinska Hospital, Stockholm, Sweden, were analyzed for the presence of immunoglobulin A (IgA) and IgG antibodies to a panel of synthetic peptides derived from the E2, L1, and L2 regions of the human papillomavirus types 1 (HPV 1), 6, 8, 11, 16, 18, 31, and 33. The study subjects were divided into three groups: (i) asymptomatic men with no history of genital warts who served as controls, (ii) men with visible condylomata, and (iii) men who had previously been afflicted with condylomata. There were no significant differences in antibody titers for any of the HPV 6- or 11-derived peptides among patients with current condylomata and the controls. For the peptide from L1 of HPV 6, there was an increase in the IgG titers among men with previous condylomata compared with the titers for the controls (52% versus 27% seropositivity; P less than 0.05). Also, for the peptide from L2 of HPV 6, there was an increase in the IgG titers among men who had been afflicted with condylomata previously (P less than 0.05). Increased IgA antibody titers against an HPV 16-derived peptide and an HPV 18-derived peptide were also detected. For the peptides from L1 and L2 of HPV 6, the study was extended to an additional group of 127 males attending the sexually transmitted disease clinic at Huddinge Hospital in southern Stockholm. Again, significantly increased antibody levels were detected only for IgG and only among asymptomatic men with a history of condylomata (P < 0.01 for the L1 peptide and P < 0.05 for the L2 peptide). The results suggest that the IgG response against the late proteins of HPV 6 reflects mainly previous exposure to the virus rather than ongoing viral disease.     

HPV31型L2保守中和表位可诱发广谱中和抗体

目的研究人乳头瘤病毒31型(HPV31)次要外壳蛋白L2保守中和表位的免疫活性及诱发抗体的中和范围。方法合成法获得HPV31 L2 aa.17-40多肽,用EDC法偶联KLH,联合弗氏佐剂免疫新西兰大白兔,用假病毒中和实验检测免疫血清对来自α4、α7、α9、α10及β1亚属的多个HPV型别的中和抗体。结果 HPV31 L2-KLH偶联肽可在新西兰大白兔体内诱发针对至少17种HPV型别的广谱中和抗体,其中HPV31的中和抗体滴度最高,HPV5/45/57的次之。结论首次发现HPV31 L2保守中和表位免疫血清具有广谱中和活性,为基于该表位的广谱HPV疫苗研发奠定了基础。     

预防性重组HPV58-减毒志贺氏杆菌载体活疫苗的研制及免疫学特性

目的：构建预防性重组HPV58-减毒志贺氏杆菌载体活疫苗，并研究其免疫学特性。方法：克隆HPV58L1基因并重组入自杀载体pCVD442，减毒志贺氏杆菌Sf301：△virG、辅助质粒PRK2013、重组自杀载体pCVD442-HPV58L1进行筛选杂交，经氨苄抗性培养基、刚果红培养基双重选择，获得重组的含HPV58L1基因的减毒志贺氏杆菌菌株。Western blot检测HPV58L1蛋白表达。用豚鼠角结膜炎模型进行疫苗动物免疫试验，ELISA检测豚鼠血清中特异性抗体，ELISPOT检测豚鼠脾及淋巴结中抗原特异性IgG、Iga产生细胞频数。结果：Western blot法证实该菌株可表达HPV58L1蛋白。豚鼠免疫保护试验发现：HPV58L1-减毒志贺氏杆菌不引起角结膜炎，经用野生型sf301攻击后有80％豚鼠不发病。免疫后第20天，免疫豚鼠血中抗HPV58L1特异性IgG、IgA明显升高，而对志贺氏杆菌菌体抗原（LPS）仅产生低效价的IgG抗体。ELISPOT结果显示，脾和淋巴结的IgA-ASC及IgG-ASC明显升高。结论：成功构建了预防性重组HPV58-减毒志贺氏杆菌载体活疫苗，能诱发免疫动物较强的体液免疫反应，激发保护性抗体的产生。     

Human Papillomavirus (HPV) L1 and L1-L2 Virus-Like Particle-Based Multiplex Assays for Measurement of HPV Virion Antibodies

Humoral immunity to human papillomavirus (HPV) has not been fully characterized, and there is currently no standard serologic test for the measurement of HPV antibodies. Most HPV serologic assays developed to date are based on virus-like particles (VLPs) of the major HPV capsid protein, L1. We sought to compare the performance of a multiplex HPV L1 VLP-based serologic assay to that of an assay based on VLPs comprised of both L1 and the minor capsid, L2. We developed HPV L1 VLP and L1-L2 VLP-based multiplex seroassays for the detection of HPV type 16 (HPV16) and HPV18 virion binding antibodies using Luminex fluorescent bead technology. We compared the performance of these assays to that of established pseudovirion-based neutralization and L1 VLP-based enzyme-linked immunosorbent assays (ELISAs). A total of 391 serum specimens from unvaccinated adult males and females were tested. The L1 and L1-L2 VLP multiplex seroassays each demonstrated substantial agreement with both the neutralization assays and the ELISAs for the detection of HPV16 antibodies (κ = 0.60 to 0.64). However, the L1-L2 VLP seroassay demonstrated better agreement with neutralization assays for the detection of HPV18 antibodies than the L1 VLP seroassay (κ = 0.74 and 0.43, respectively). L1 and L1-L2 VLP seroassays showed excellent agreement with one another for the detection of HPV16 antibodies (κ = 0.86) but only moderate agreement for HPV18 antibodies (κ = 0.44). The HPV L1-L2 VLP seroassay performs well for the concurrent measurement of HPV16 and -18 antibodies in large numbers of samples and may be extended to include other HPV types.     

Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection

Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.     

Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virion-like particles.

Mice of three haplotypes (H-2d, H-2b, and H-2d/b) were immunized with synthetic HPV16 virus-like particles (VLPs), produced using a vaccinia virus doubly recombinant for the L1 and L2 proteins of HPV16. The resultant anti-VLP antisera recognized HPV16 capsids by ELISA assay and baculovirus recombinant HPV16 L1 and L2 protein on immunoblot. Overlapping peptides corresponding to the HPV16 L1 amino acid sequence were used to define the immunoreactive regions of the L1 protein. The majority of the L1 peptides were reactive with IgG from the mice immunized with the synthetic HPV16 capsids. A computer algorithm predicted seven B epitopes in HPV16 L1, five of which lay within peptides strongly reactive with the murine antisera. The murine anti-VLP antisera failed to react with the two peptides recognized by anti-HPV16L1 monoclonal antibodies raised by others against recombinant L1 fusion protein. We conclude that the immunoreactive epitopes of HPV16 defined using virus-like particles differ significantly from those defined using recombinant HPV16 L1 fusion proteins, which implies that such fusion proteins may not be the antigens to look for HPV16L1 specific immune responses in HPV-infected patients.     

Development and evaluation of a colorimetric PCR system for the detection and typing of human papillomaviruses

In developing countries, the introduction of human papillomaviruses (HPV) DNA testing as an adjunct to cytological screening programs has been delayed due to the lack of high performance and cost effective diagnostic nucleic acid methods. In this study we report the development and evaluation of the L1HPVPCR, a PCR-based method for the detection and typing of five of the most prevalent high-risk HPV types. The L1HPVPCR assay combines amplification with the MY09/11 HPV consensus primer system, liquid hybridization of the PCR products with no radioactive probes and enzyme immunoassay analysis. The technique is a user-friendly system that allows accurate HPV DNA detection and typing with inexpensive instrumentation that could be performed with not sophisticated reagents in almost any laboratory. Different cutoff points for generic and specific HPV detection were determined using reproducibility analysis and receiver operating characteristic curves to ensure good analytical sensitivity and clinical effectiveness. We used the L1HPVPCR assay to estimate the prevalence of HPV infection in 127 women at risk of cervical cancer from the city of Rosario (Argentina), where no epidemiological data has been previously reported. Further, we explored the clinical utility of the L1HPVPCR assay respect the Pap smear using a combined diagnosis of cytology, histology and colposcopy as gold standard. In conclusion, our results indicate that the assay described here provides a tool for accurate HPV DNA testing and could be applied in regions where no commercial tests are available.