Polymer-based enzyme-linked immunosorbent assay using human papillomavirus type 16 (HPV16) virus-like particles detects HPV16 clade-specific serologic responses

Human papillomavirus type 16 (HPV16) virus-like particles (VLP) were used as antigen in a polymer enzyme-linked immunosorbent assay (ELISA) to measure antibodies to HPV capsid proteins. Serum samples from 575 college women, previously tested for the presence of cervicovaginal HPV DNA, were analyzed. The prevalences of anti-HPV16 VLP antibodies at baseline were 14.1% for immunoglobulin G (IgG) and 6.4% for IgA. The seroprevalences of IgG in women with cervicovaginal HPV16, HPV16-related types, and other HPV types were 55, 33, and 19%, respectively (P < 0.001), compared to the prevalence in women without an HPV infection (10%). HPV VLP IgA seropositivity was associated with high HPV16 VLP IgG optical density values. The seropositivity of IgG antibodies was independently associated with infection with HPV16 or HPV16-related types, increased number of lifetime male partners for vaginal sex, having sex with men >/= 5 years older, history of abnormal PAP smear, older age, and living separately from parents. Use of HPV16 VLP polymer ELISA detects clade-specific responses and suggests an HPV16 VLP vaccine may have broader protection that initially anticipated.     

Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice.

Attenuated strains of Salmonella are attractive live vaccine candidates for eliciting mucosal as well as systemic immune responses. The ability to induce immune responses in the reproductive tract may be critical for the effectiveness of a prophylactic vaccine against genital human papillomaviruses (HPV), which are important etiologic agents in the development of cervical cancer. To examine the potential of a live Salmonella-based vaccine to prevent genital HPV infection, the L1 major capsid protein from HPV type 16 (HPV16) was constitutively expressed in the PhoPc strain of Salmonella typhimurium. As demonstrated by electron microscopy, the L1 protein expressed in these bacteria assembled into virus-like particles (VLPs) that resemble authentic papillomavirus virions. This is the first demonstration that papillomavirus VLPs can self-assemble in prokaryotes. BALB/c mice were immunized with the HPV16 L1 recombinant PhoPc strain by the oral and nasal routes. Despite a low stability of the L1-expressing plasmid in vivo, a double nasal immunization was effective in inducing L1-specific serum antibodies that recognized mainly native, but not disassembled, VLPs. These antibodies effectively neutralized HPV16 pseudotyped virions in an in vitro infectivity assay. Conformationally dependent anti-VLP immunoglobulin A (IgA) and IgG were also detected in oral and vaginal secretions, indicating that potentially protective antibody responses were elicited at mucosal sites. Recombinant attenuated Salmonella expressing HPV capsids may represent a promising vaccine candidate against genital HPV infection.     

表达野生型和突变型HPV16-E7重组痘苗病毒诱发的抗肿瘤免疫反应

目的 选出适合于治疗性疫苗研制的HPV16E7突变基因。方法 对表达野生型和突变型E7蛋白的重组痘苗病毒所诱发的细胞免疫反应和抗肿瘤活性进行比较研究。结果 表达突变型ME7-1（24G26G）的重组痘苗病毒VmE7-1与表达野生型E7的VwE7相同,可诱发特异性抗体和CTL的产生,明显推迟成瘤时间并且保护部分小鼠抵抗肿瘤细胞的攻击;而表达突变型ME7-2（24G26G91G）的重组痘苗病毒VmE7-2免疫小鼠后难以有效的激发细胞免疫反应,在抗肿瘤移植实验中也不具明显的免疫保护作用。结论 E7突变基因ME7-1可作为候选基因用于HPV16治疗性疫苗的研制。     

Prevalence of antibodies to human papillomavirus (HPV) type 16 virus-like particles in relation to cervical HPV infection among college women.

A human papillomavirus type 16 (HPV-16) virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA) was used to measure serum antibody to capsid proteins in 376 sexually active college women who were also screened for the presence of genital HPVs by PCR and interviewed for demographic and behavioral risk factors for HPV infection. The seroprevalence was 46% in women with HPV-16 DNA in the genital tract. The corresponding values for women who harbored other HPV types or no HPV in the genital tract were 30 and 19%, respectively (HPV-16 group versus no-HPV group; odds ratio [OR], 3.7; 95% confidence interval [CI], 1.5 to 8.9). The antibody response was significantly higher among women with a high viral load than among those with a low viral load (median optical density value, 0.838 versus 0.137, P = 0.009). Comparable levels of seroreactivity were observed among women infected with HPV types distantly or closely related genetically to HPV-16. Seroreactivity was significantly associated with an age of 25 to 30 years (OR, 2.3; 95% CI, 1.2 to 4.4), three or more lifetime sexual partners (OR, 2.9; 95% CI, 1.1 to 10), and history of a sexually transmitted disease other than HPV (OR, 3.1; 95% CI, 1.5 to 6.3). The percent seropositivity increased linearly with number of lifetime sexual partners until reaching a plateau at 35% for women with more than six partners (chi for linear trend, P < 0.001). The low sensitivity of HPV-16 VLP-based ELISA may limit the usefulness of the assay as a diagnostic test for HPV-16 infection. However, the assay appears to have adequate specificity and should be useful as an epidemiological marker of HPV-16 infection and sexual behavior.     

Cross-reactivity among several recombinant calicivirus virus-like particles (VLPs) with monoclonal antibodies obtained from mice immunized orally with one type of VLP

Human caliciviruses (HuCVs) are classified into the Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV) as genera within the family CALICIVIRIDAE: The NLV genus is further classified into genogroups I and II, based on sequence similarities. To study the antigenic determinants on the HuCV capsid protein and develop new diagnostic tools for field samples, we established and characterized monoclonal antibodies (MAbs) against baculovirus-expressed recombinant HuCV virus-like particles (VLPs). Hybrid clones producing MAbs were obtained from cultures of PAI myeloma cells fused with spleen or mesenteric lymph node cells from mice immunized orally with either a single type of recombinant Norwalk virus (rNV), Kashiwa 47 virus (rKAV), Snow Mountain agent (rSMA), or Sapporo virus (rSV) VLP or with mixtures of two types of VLPs from different genogroups. Twenty MAbs, obtained as mouse ascites, were characterized and classified into six groups according to their enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) cross-reactivity patterns to VLPs. Five groups of MAbs reacted by both WB and ELISA and were classified as follows: common cross-reactive MAbs for four genogroup I and six genogroup II VLPs (group A), genogroup I-specific MAbs (group B), genogroup II-specific MAbs (group C), and strain-specific MAbs (groups D and E). One MAb group (group F) reacted only by ELISA. The group A MAbs, which showed broad cross-reactivity with VLPs of both NLV genogroups, were obtained from mice immunized orally with a single type of VLP (either rNV or rKAV). Two MAbs, which were obtained from mice immunized with rSV, reacted with rSV but not with any NLV VLP. These are the first MAbs to be reported for any SLV. These strain-, genogroup-, and genus-reactive MAbs will be useful tools for further study of the antigenic and structural topography of the HuCV virion and for diagnostic assays for HuCVs.     

Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts.

Thirty-nine patients with condylomas (12 women and 27 men) attending a dermatology clinic were tested for genital human papillomavirus (HPV) DNA and for seroprevalence to HPV type 6 (HPV6) L1 virus-like particles. The L1 consensus PCR system (with primers MY09 and MY11) was used to determine the presence and types of HPV in sample specimens. All 37 (100%) patients with sufficient DNA specimens were positive for HPV DNA, and 35 (94%) had HPV6 DNA detected at the wart site. Three patients (8%) had HPV11 detected at the wart site, and one patient had both HPV6 and -11 detected at the wart site. Thirteen additional HPV types were detected among the patients; the most frequent were HPV54 (8%) and HPV58 (8%). Baculovirus-expressed HPV6 L1 virus-like particles were used in enzyme-linked immunosorbent assays to determine seroprevalence among the patients with warts. Seronegativity was defined by a control group of 21 women who were consistently PCR negative for HPV DNA. Seroprevalence was also determined for reference groups that included cytologically normal women who had detectable DNA from either HPV6 or HPV16 and women with HPV16-associated cervical intraepithelial neoplasia. Among the asymptomatic women with HPV6, only 2 of 9 (22%) were seropositive, compared with 12 of 12 (100%) female patients with warts. A similar trend in increased HPV6 seropositivity with increased grade of disease was found with the HPV16 DNA-positive women, whose seroprevalence increased from 1 in 11 (9%) in cytologically normal women to 6 in 15 (40%) among women with cervical intraepithelial neoplasia 1 or 3. However, only 4 of 25 (16%) male patients were seropositive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of antibodies against human papillomavirus (HPV) type 16 virions by enzyme-linked immunosorbent assay using recombinant HPV 16 L1 capsids produced by recombinant baculovirus.

The L1 major capsid protein of human papillomavirus type 16 (HPV-16) was expressed in Sf-21 insect cells with a recombinant baculovirus. Virus-like particles obtained were purified and used to develop an enzyme-linked immunosorbent assay for detection of anti-HPV-16 antibodies in sera from 76 women with evidence of genital HPV infection and 79 controls. HPV-16-infected individuals developed antibodies directed at HPV-16 virions since reactivity against recombinant HPV-16L1 capsids was observed in 50% of them compared with only 6% in the general adult population. However, some cross-reactivities with sera from women infected with others HPV types were observed.     

人乳头瘤病毒16型野毒株L1-E7融合基因重组腺病毒载体构建及在293细胞中表达嵌合病毒样颗粒的研究

目的制备人乳头瘤病毒HPV16L1-E7重组腺病毒,以期获得防治宫颈癌的重组腺病毒减毒活疫苗。方法以HPV16型的野毒株HPV16-114/K为模板,利用PCR克隆技术制备HPV16L1-E7融合基因pUC19L1-E7,含L1的1～301氨基酸（AA）和E7的1～60AA的编码DNA序列;并转入腺病毒穿梭质粒pCA14L1-E7,与腺病毒质粒pBHG10共转染293细胞,筛选重组腺病毒rAd5HPV16L1-E7。以PCR、ELISA和Westernblot方法鉴定重组病毒及其表达的L1-E7蛋白,密度梯度超速离心纯化HPV16嵌合L1-E7VLP,电镜观察VLP的形态结构。结果PCR-DNA序列分析表明成功构建了HPV16L1-E7重组质粒pUC19L1-E7;并获得HPV16重组腺病毒rAd5HPV16L1-E7,在293细胞中可高效表达L1-E7蛋白,并形成嵌合病毒样颗粒（cVLP）。结论构建的重组腺病毒rAd5HPV16L1-E7可有效表达HPV16L1-E7cVLP,可进一步用于动物实验,研究其在防治宫颈癌中的作用。     

Immunization against human papillomavirus type 16 tumor cells with recombinant vaccinia viruses expressing E6 and E7.

Papillomaviruses are etiological agents of epithelial proliferative disease. In man, neoplastic transformation of the uterine cervix has been linked to infection with specific subtypes of human papillomavirus, particularly types 16 and 18. We previously reported that live vaccinia virus recombinants expressing early transforming proteins of other tumor viruses can immunize against challenge with cognate tumor cells and we have extended this approach to HPV16. Neoplastic transformation by papillomaviruses involves expression of early open reading frames (ORFs) E5, E6, and E7, and we report the construction of vaccinia recombinants separately expressing ORFs E5-E7 of HPV16. Primary rat cell lines cotransformed with HPV16 and an activated ras oncogene were established in order to evaluate the potential of the recombinants to elicit antitumor immunity. We report that inoculation of rats with vaccinia recombinants expressing E6 or E7 retarded or prevented tumor development in a proportion of animals challenged by subcutaneous seeding of tumor cells whereas the recombinant expressing E5 was inactive.     

人乳头瘤病毒16型结构基因L1在昆虫细胞中的表达及其生物学活性

目的 研究与宫颈癌及人类其它多种组织癌症密切相关的人乳头瘤病毒16型（HPV16）的晚期基因L1的表达及其生物学活性。方法 采用PCR法从pBSSK-B/16L1扩增了来自中国妇女鲍温病组织标本的HPV16晚期基因L1,装入杆状病毒转移载体;在DH10Bac内通过转座子Tn7的介导,使携带有杆状病毒多角体蛋白基因启动子Ppolh的HPV16 L1基因整合入杆状病毒,形成重组杆状病毒;转染昆虫细胞Sf9进行表达;将感染72h的Sf9细胞包埋、切片、染色后,电镜观察。提取L1蛋白,免疫BALB／ c小鼠。结果 重组杆状病毒在Sf9细胞内高效表达出L1蛋白,经Western blot发现能与L1抗体特异地结合;薄层扫描显示所表达的L1蛋白占Sf9细胞总蛋白的比例高达31％。经透射电镜观察表明,在细胞核里有大量的重组杆状病毒形成;并且产生了大量的由HPV16 L1蛋白单体自组装的病毒样颗粒（virus-like particles,VLP)。小鼠免疫实验结果表明,所表达的HPV16 L1蛋白单体自组装的病毒样颗粒（virus-like particles,VLP)。小鼠免疫实验结果表明,所表达的HPV16 L1蛋白具有强免疫原性。结论 此研究为今后L1基因分子流行病学检查、L1蛋白的结构生物学研究、疫苗研制以及HPV相关基础性研究提供了有用的资料。     

人乳头状瘤病毒16型结构蛋白L1和L2在大肠埃希菌 …

目的　研究人乳头状瘤病毒(HPV)主要结构蛋白L1(HPV16L1)和次要结构蛋白L2(HPV16L2)在原核系统的表达。方法　采用pET30a载体分别构建pET30aL1和pET30aL2质粒,并分别转入大肠埃希菌BL21菌株,经IPTG(异丙基硫代βD半乳糖苷)诱导,表达融合蛋白6×HisL1和6×HisL2,用SDSPAGE和Westernblot方法进行检测。结果　6×HisL1和6×HisL2融合蛋白在BL21菌中高效表达,约2～3mgml,其相对分子质量分别约为60000和97000;6×HisL1降解较6×HisL2多。结论　HPV16结构蛋白L1和L2能在原核表达系统高效表达;6×HisL2稳定性高于6×HisL1融合蛋白。     

Memory T cells specific for novel human papillomavirus type 16 (HPV16) E6 epitopes in women whose HPV16 infection has become undetectable

Human papillomavirus (HPV)-specific T-cell response to the HPV type 16 (HPV16) E6 protein has been shown to be associated with successful viral clearance. The patterns of CD8 T-cell epitopes within HPV16 E6 protein were previously studied in two women with HPV16 clearance. The goal of this study was to characterize these epitopes in terms of their minimal and optimal amino acid sequences and the human leukocyte antigen (HLA) restriction molecules. The presence of the epitope-specific memory T cells after viral clearance was also examined. In subject A, the dominant epitope was characterized to be E6 75-83 (KFYSKISEY), restricted by the HLA-B62 molecule, while that of subject B was E6 133-142 (HNIRGRWTGR), restricted by the HLA-A6801 molecule. Homologous epitopes were identified in five other high-risk HPV types for both of these epitopes, but they were not recognized by respective T-cell clone cells. An enzyme-linked immunospot assay or tetramer analysis was performed on peripheral blood mononuclear cells from blood samples collected after viral clearance but prior to isolation of the T-cell clones. The presence of epitope-specific memory T cells was demonstrated. These data suggest that HPV-specific memory T cells were generated in vivo and that they may remain in circulation many months, if not years, after viral clearance. Our findings broaden the spectrum of the CD8 T-cell epitopes of the HPV16 E6 protein. The characterization of novel T-cell epitopes and long-lasting epitope-specific memory T cells may be useful for the development of a potential epitope-based vaccine.     

Analysis of human papillomavirus type 16 (HPV16) DNA load and physical state for identification of HPV16-infected women with high-grade lesions or cervical carcinoma

Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 10³ cells in normal samples up to 56,354 copies per 10³ cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/10³ cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.     

Codon optimization of the human papillomavirus 11 (HPV 11) L1 gene leads to increased gene expression and formation of virus-like particles in mammalian epithelial cells

The 505 amino acid L1 protein of the human papillomavirus type 11 (HPV 11) is the major capsid polypeptide that has been shown to self-assemble into virus-like particles (VLPs) in vivo and in vitro. While L1 is essential for viral infection, expression studies in mammalian cells have been hampered by different codon preference between the virus and its host. To optimize L1 gene expression in mammalian cells, we converted wild-type HPV 11 L1 (11 L1wt) codons to those more common in human genes. The modified HPV 11 L1 gene (11 L1h) generated protein levels that were at least 100-fold higher than those of wild-type HPV 11 L1, while no obvious differences were seen in the level of mRNA. HPV 11 L1 protein was detected in mammalian epithelial and fibroblast cells, by immunoblotting and indirect immunofluorescence (IIF) techniques. Unlike the situation in situ, IIF revealed the presence of L1 mainly at perinuclear sites. Virus-like particles assembled intranuclearly only to a low extent, as indicated by transmission electron microscopy. DNA vaccination using the HPV 11 L1h gene yielded a drastic increase in L1-specific antibody production in mice as compared to immunization with the wild-type gene.     

稳定表达人乳头瘤病毒(HPV)58型E6E7融合基因的B16细胞系的建立

目的:构建pIRES-neo-HPV58E6E7真核表达载体,稳定转染入小鼠黑色素瘤B16细胞,建立稳定表达HPV58E6E7基因的B16细胞系。方法:采用PCR方法扩增出HPV58E6E7融合基因的全长序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo中,并加入酶切位点和6×his标签,构建重组真核表达质粒pIRES-neo-HPV58E6E7。利用阳离子脂质体介导法将其转染入小鼠黑色素瘤B16细胞,经G418加压筛选出稳定转染的阳性克隆。利用Western blot、流式细胞术、免疫荧光等检测方法验证HPV58E6E7融合基因在稳定转染的B16细胞株中的表达。结果:经PCR、限制性内切酶鉴定及测序分析,pIRES-neo-HPV58E6E7重组质粒构建正确,转染B16细胞株后,经过Western blot、流式细胞术和免疫荧光等检测显示B16细胞株能够稳定、高效表达HPV58E6E7融合基因,表明B16-HPV58E6E7稳定转染细胞系构建成功。结论:成功构建了pIRES-neo-HPV58E6E7真核表达载体,建立了可以高效、稳定表达HPV58E6E7融合基因的B16细胞系。该稳定转染细胞系的建立为进一步研究HPV58治疗性基因疫苗的功能提供了良好的靶细胞,为其在肿瘤免疫治疗中的应用奠定了研究基础。     

Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically na?ve to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of E6-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised E6-transgenic mice that have not developed inflammatory skin disease remain immunologically na?ve to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines.     

Antileukemic effects of recombinant human tumor necrosis factor alpha (rh-TNF alpha) with cyclophosphamide or methotrexate on leukemia L1210 and leukemia P388 in mice.

We investigated the influence of recombinant human tumor necrosis factor alpha (rh-TNF alpha) administered as a single agent or in combination with cyclophosphamide (CY) or methotrexate (MTX) on the survival time of mice inoculated with lymphoblastic leukemia L1210 or lymphatic leukemia P388. The median survival time of leukemia L1210 bearing mice treated with rh-TNF alpha at doses ranging from 200 to 275 g/kg in daily i.p. injections was longer than that of control animals. Groups of mice with leukemia L1210 receiving rh-TNF alpha combined with either MTX or CY lived longer than animals treated with these agents individually. We observed only slight prolongation of life of animals inoculated with this tumor and treated with rh-TNF alpha at dose of 800 micrograms/kg in four injections on 2, 4, 6 and 8 day of experiment, and no effect when rh-TNF alpha was administered at dose of 200 or 400 micrograms/kg at the same treatment regime. In contrast no significant differences in lifetime were obtained from either simultaneous or sequential treatment of mice bearing leukemia P388. Groups of mice with this tumor treated with rh-TNF alpha in conjunction with either MTX or CY lived longer than controls, or rh-TNF alpha singly treated mice, but their survivals were not significantly prolonged compared with mice receiving cytostatics alone.     

Oral immunization of interleukin-4 (IL-4) knockout mice with a recombinant Salmonella strain or cholera toxin reveals that CD4+ Th2 cells producing IL-6 and IL-10 are associated with mucosal immunoglobulin A responses.

Mucosal immunoglobulin A (IgA) responses are often associated with Th2-type cells and derived cytokines, and interleukin-4 (IL-4) knockout (IL-4-/-) mice with impaired Th2 cells respond poorly to oral antigens. However, we have noted that IL-4-/- mice have normal mucosal IgA levels, which led us to query whether different oral delivery systems could elicit mucosal immunity. Two oral regimens were used: (i) a live recombinant Salmonella strain which expresses fragment C (ToxC) of tetanus toxin, and (ii) soluble tetanus toxoid (TT) with cholera toxin (CT) as an adjuvant. Oral immunization of IL-4-/- mice with recombinant Salmonella vaccine expressing ToxC induced brisk mucosal IgA and serum IgG (mainly IgG2a) anti-TT antibody responses. TT-specific CD4+ T cells from spleen or Peyer's patches produced gamma interferon, indicative of Th1 responses; however, IL-6 and IL-10 were also seen. Oral immunization of IL-4-/- mice with TT and CT induced weak mucosal IgA to TT; however, brisk IgA anti-CT-B responses and CT-B-specific CD4+ T cells producing IL-6 and IL-10 were also noted. These results show that although IL-4-dependent antibody responses are impaired, mucosal IgA responses are induced in IL-4-/- mice. These result suggest that certain cytokines, i.e., IL-6 and IL-10 from Th2-type cells, play an important compensatory role in the induction and regulation of mucosal IgA responses.