Detection of human papillomavirus DNA in cervical cancer tissue by the polymerase chain reaction

A method for detecting HPV DNA in cervical cancer tissue was developed without using isotopes. The DNA samples from the cancer tissues were first subjected to amplification by PCR, followed by polyacrylamide gel electrophoresis to identify the specific amplified fragment. The specificity and sensitivity of the PCR method are described. Compared with the dot hybridization technique, it is shown that the method is able to detect HPV DNA in cervical cancer tissues.     

Polyoma virus-transformed rat cell lines inducible for viral capsid antigen synthesis.

A number of polyoma virus (PV)-transformed lines of rat, mouse and hamster origin established in the course of this study were investigated for virus induction by mitomycin C. Neither infectious virus nor V-antigen was detected in six mouse and four hamster lines. Out of 14 rat lines, three were found inducible for infectious virus, and one could be induced only for V-antigen synthesis. Two lines, designated as RPA and RPB differ from the others with respect to their response to mitomycin C and elevated temperature (40°). The proportion of V-antigen-containing cells increased 50- and 60-fold in the RPA and RPB lines, respectively, when the mitomycin C-treated cultures were subsequently incubated at 40° for 24 hr, as compared to the cultures kept continuously at 37°. No infectious virus was detected in the mitomycin C-treated RPA cultures incubated at either temperature. The RPB line can be induced for infectious virus production at a cell frequency of $\text{1}\text{100}$ V-antigen-containing cells when incubated at 37°. Though the proportion of V-antigen-containing cells increased in the heated cultures of this line 60-fold, the number of plaques produced by the cell extracts and the proportion of virus-producing cells were similar both in the heated and unheated cultures. Fifty percent of clones isolated from the RPA line and about 80% of RPB clones are inducible, exhibiting, except for one RPB clone, a similar pattern of response to the inducing agents as the parental lines. In heterokaryon cultures of untreated mouse embryo cells and RPA or RPB cells pretreated with mitomycin C and heat, a proportion of multinucleated cells was shown to contain V-antigen. However, virus maturation was not detected in the heterokaryon cultures with RPA cells and was not significantly enhanced in the cultures with RPB cells. We also studied the effect of elevated temperature (40°) on PV induction by mitomycin C in highly inducible PV-transformed lines. In contrast to the RPA and RPB lines, in the highly inducible lines, the elevated temperature, though exerting a relatively weak enhancing effect on induction by mitomycin C, increased the proportion of V-antigen-containing cells and the virus yields to an almost equal extent. These results would indicate that the lack of virus production in the V-antigen-containing RPA and in most RPB cells is due to a deficiency of the viral genome persisting in these cells and that a temperature-sensitive factor is involved in the process of V-antigen induction in these lines.     

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.     

Epstein-Barr病毒壳抗原的纯化及其在检查血清抗体中的应用

为了建立纯化Ｅｐｓｔｅｉｎ－Ｂａｒｒ（ＥＢ）病毒壳抗原（ＥＢＶ－ＶＣＡ）的方法，用于酶联免疫吸附试验（ＥＬＩＳＡ）检测人血清中的相关抗体，我们用重组昆虫病毒在感染的ｓｆ９细胞中表达ＥＢＶ－ＶＣＡ。感染的细胞经裂解后，层析纯化表达产物。用纯化的ＶＣＡ作为抗原包被ＥＬＩＳＡ板或硝基纤维膜，检查血清中ＶＣＡ／ＩｇＡ和ＶＣＡ／ＩｇＧ抗体，为ＥＢＶ感染的检测和ＮＰＣ的诊断发展了一个敏感、特异和简便的方法。     

Epstein—Barr病毒壳抗原的纯化及其在检查血清抗体…

为了建立纯化Ｅｐｓｔｅｉｎ－Ｂａｒｒ（ＥＢ）病毒壳抗原（ＥＢＶ－ＶＣＡ）的方法，用于酶联免疫吸附试验（ＥＬＩＳＡ）检测人因清中的相关抗体，我们用重组昆虫病毒在感染的ｓｆ９细胞中表达ＥＢＶ－ＶＣＡ。感染的细胞经裂解后，层析纯化表达产物。用经的ＶＣＡ作为抗原包虫被ＥＬＩＳＡ板或硝基纤维膜，检查血清中ＶＣＡ／ＩｇＡ和ＶＣＡ／ｇＧ抗体，为ＥＢＶ感染的的检测和ＮＰＣ的诊断发展了一个敏感、特异和简便的方法。     

Cloning and expression of two human recombinant monoclonal Fab fragments specific for EBV viral capsid antigen

Serum titers of antibody to Epstein-Barr virus (EBV) viral capsid antigen (VCA) have been positively correlated with malignancies of lymphoid proliferation, such as Burkitt's lymphoma and Hodgkin's lymphoma. We have constructed a phage display combinatorial antibody Fab library from a patient with marginal zone B cell lymphoma associated with Sj?gren's syndrome and carrying high serum anti-EBV-VCA IgG titer. Fab fragments were selected by panning against EBV-VCA protein coated onto ELISA plates, and selected Fab clones were characterized by ELISA, western blotting (WB), indirect immunofluorescence assay and immunohistochemistry. We have established two Fab clones, Fab-aVCA1 and Fab-aVCA21, which specifically recognize EBV-VCA by ELISA and WB. Inhibition ELISA competition showed that both clones could significantly reduce the binding of specific anti-EBV-VCA mAb to its relevant proteins. Furthermore, these two Fab clones could localize VCA protein in the EBV-positive P3HR1 and Daudi cell lines, as well as in tissue samples from patients with EBV-infected lymphoid malignancies. These results indicate that our two Fab clones are novel human mAbs specific for EBV-VCA protein and may have potential benefits for development of novel diagnostic and therapeutic approaches in EBV-related lymphoid malignancies.     

Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history.

BACKGROUND: Recent studies have revealed a possible role for the human papillomavirus (HPV) in the pathogenesis of breast cancer. In this study, patients having both a history of invasive cervical cancer and breast cancer as second primary cancer were selected for enrolment in a study of breast carcinomas for the presence of HPV. METHODS: Paraffin-embedded tissue from cervical cancer, pelvic lymph nodes, breast cancer and axillary lymph nodes of eleven patients were examined for the presence of HPV DNA using a polymerase chain reaction - enzyme immuno assay. DNA extraction was performed with the "QIAamp Tissue Kit" according to the manufacturer's instructions. Additionally, serum samples taken between diagnosis of cervical and breast cancer, were analyzed for the presence of HPV DNA to examine a possible haematogenous spread of oncogenic HPV DNA. RESULTS: All cervical carcinomas were HPV-positive. HPV DNA was detected in seven out of eleven cases in breast cancer and/or axillary lymph node tissue. Six patients had the same HPV type (HPV-16) in cervical cancer and in the corresponding breast cancer/lymph node tissue. In one case, the same HPV DNA type (HPV 16) was detected in cervical cancer, breast cancer and serum sample. CONCLUSION: These results suggest that HPV DNA might be transported from the original site of infection to the breast tissue by the bloodstream, and that it is possibly involved in the carcinogenesis of breast neoplasia in some patients.     

Yeast coexpression of human papillomavirus types 6 and 16 capsid proteins

The L1 and L2 capsid proteins of animal and human papillomaviruses (HPVs) can self-assemble into virus-like particles (VLPs) that closely resemble native virions. The use of different animal models shows that VLPs can be very efficient at inducing a protective immune response. However, studies with infectious HPV virions and VLPs of different HPV types indicate that the immune response is predominantly type-specific. We have generated a diploid yeast strain that coexpresses the L1 and L2 capsid proteins of both HPV-6b and HPV-16, and we have purified fully assembled VLPs banding in a cesium chloride gradient at the expected density of 1.29-1.3 mg/ml. Experimental evidence strongly indicated that the four proteins coassembled into VLPs. Western blot analysis, using anti-HPV-6 and anti-HPV-16 L1-specific monoclonal antibodies and type-specific L2 antisera, demonstrated that all four proteins copurified. Most importantly, immunoprecipitation experiments, carried out using type-specific anti-L1 monoclonals and either total yeast cell extracts or purified VLPs, confirmed the interaction and the formation of covalent disulfide bonds between the two L1 proteins. Finally, HPV-6/16 VLPs administered to mice induced conformational antibodies against both L1 protein types. These results suggest that coexpression of different capsid proteins may provide new tools for the induction of antibodies directed against multiple HPV types.     

Epstein-Barr virus (P3HR-1) defective DNA codes for components of both the early antigen and viral capsid antigen complexes

A set of lambda phages containing overlapping fragments of Epstein-Barr virus (EBV) defective DNA has been cloned from P3HR-1-superinfected Raji cells. Mapping data obtained using these cloned DNA fragments confirmed the structure of P3HR-1 defective DNA previously deduced directly from virion DNA (M.-S. Cho, G. W. Bornkamm, and H. zur Hausen, 1984, J. Virol., in press). The ability of the cloned defective DNA fragments to induce EBV antigens in transfected baby hamster kidney (BHK) cells was tested using indirect immunofluorescence assays. Up to 5% of those cells receiving a defective DNA fragment BamHI-W'C' transiently expressed a de novo nuclear antigen which was identified as being a component of the EAD complex by its reactivity with characterized EBV-positive human sera. A 20-kb clone of P3HR-1 defective DNA (EcoRI-C1) was found to induce the synthesis of a component of the VCA complex. One percent of cells transfected with this clone showed cytoplasmic fluorescence when tested with either VCA+ human sera or EBV anti-VCA monoclonal antibody. Subcloning of the EcoRI-C1 fragment localized the VCA gene to a 4.1-kb segment which maps within the BamHI-A fragment of the standard genome. This segment contains a single large open reading frame of 2.6 kb (B. Barrell, A. Bankier, R. Baer, P. Biggin, P. Deininger, P. Farrell, T. Gibson, G. Hatfull, G. Hudson, S. Stachwell, and C. Sequin, 1984, Nature (London), in press). None of the defective DNA clones were capable of inducing EBV-specific nuclear antigens (EBNAs) which is consistent with the absence of the known EBNA coding regions from the defective genome.     

Immunohistochemical demonstration of papillomavirus structural antigen in urinary bladder papillary tumors

Clear cells with characteristic features of koilocytotic atypia (perinuclear clear zone, peripheral cytoplasmic condensation and hyperchromatic nuclei), were found in 35 out of 744 urothelial papillary tumors (UPT) of the human bladder. Using a peroxidase-antiperoxidase staining technique, papillomavirus common structural antigen was detected in six of the samples studied. The results suggests that urinary bladder tumors like some other genital cancer, may be linked to papillomavirus infection.     

Detection of capsid antigen of human papillomavirus (HPV) in benign lesions of female genital tract using anti-HPV monoclonal antibody.

We established a murine monoclonal antibody (K1H8) to human papillomavirus (HPV) using alkaline-disrupted virions of HPV type 1 (HPV-1) as the immunogen. K1H8 recognized a 57 kD capsid protein of HPV-1 and detected the antigen in paraffin sections of formalin-fixed tissue. With K1H8, we examined immunohistochemically 68 biopsy specimens obtained from the female genital tract. The specimens were histologically condyloma acuminatum or koilocytotic lesions with or without dysplasia and each specimen was found to harbour a single type of genital HPV, such as types 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, and 58, by Southern blot hybridization analysis. The antigen was localized in the nuclei and occasionally in the cytoplasm of squamous cells showing koilocytotic changes. Eighty-four per cent of the specimens (57 cases) showed positivity for the antigen, indicating that K1H8 is a broadly-reactive antibody to various genital HPVs. The results suggest that benign mucosal lesions of the female genital tract are more frequently associated with viral production and are a potential source of transmission.     

Therapeutic human papillomavirus DNA vaccination strategies to control cervical cancer

A persistent human papillomavirus (HPV) infection is considered causal and necessary for the continued growth of cervical cancer. Thus, vaccination against HPV represents a plausible approach to prevent and treat cervical cancer. A report in the current issue of the European Journal of Immunology describes a therapeutic HPV DNA vaccination strategy using the HPV-16 E7 antigen fused to the invariant chain to enhance the E7-specific CD8+ and CD4+ T cell immune responses, resulting in a potent anti-tumor effect against E7-expressing tumors. Continued exploration of HPV therapeutic DNA vaccines may lead to eventual clinical application.     

Functional assessment and structural basis of antibody binding to human papillomavirus capsid

Persistent high‐risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus‐like particle (VLP)‐based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)‐based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV‐based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D‐2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher‐resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In‐depth understanding of epitope composition would ensure the development of function‐indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type‐specific epitopes would enable rational design of cross‐type neutralization via epitope re‐engineering or epitope grafting in hybrid VLPs. Copyright © 2015 John Wiley & Sons, Ltd.     

Psychosocial modulation of antibody to Epstein-Barr viral capsid antigen and human herpesvirus type-6 in HIV-1-infected and at-risk gay men.

We investigated the effects of two behavioral interventions--aerobic exercise and cognitive behavioral stress management (CBSM)--on Epstein-Barr virus viral capsid antigen (EBV-VCA) and human herpesvirus type-6 (HHV-6) antibody modulation in 65 asymptomatic gay men measured at several time points in the 5 weeks preceding and following notification of their human immunodeficiency virus-type 1 (HIV-1) serostatus. After accounting for potential immunomodulatory confounds, we found that HIV-1 seropositive men had higher EBV-VCA antibody titers than those diagnosed as seronegative at every time point during the study; however, no significant differences were found with respect to HHV-6. Among HIV-1 seropositive and seronegative subjects, respectively, those randomized to either behavioral intervention had significant decreases in both EBV-VCA and HHV-6 antibody titers over the course of the intervention as compared with assessment-only controls (of HIV-1 seropositive and seronegative status) whose antibody titers did not significantly change and which remained consistently higher than either serostatus-matched intervention group over subsequent time points, independent of total immunoglobulin G levels and degree of polyclonal B cell activation. In attempting to explain serostatus differences in EBV and HHV-6 values, it was found that HIV-1 seropositive men had significantly lower CD4 cells, CD4:CD8 ratio, and blastogenic response to phytohemagglutinin (PHA), as well as significantly higher CD8 cells at baseline. No significant differences were found between the HIV-1 seropositive and seronegative men with respect to anxiety and depression at baseline. Since the greatest changes in EBV and HHV-6 occurred between baseline and week 10, we correlated changes in immune (CD4, CD8, CD4:CD8 ratio, PHA stimulation) and distress-related markers (state depression and anxiety) with EBV and HHV-6 change scores over this time period. No significant correlations were found between any of these immune- or distress-related variable and the antibody change scores suggesting that the mechanisms by which EBV and HHV-6 antibodies are being modulated by these interventions possibly involve other, yet to be determined, immune, neuroendocrine, and/or psychologic variables.     

Emotional repression, stress disclosure responses, and Epstein-Barr viral capsid antigen titers

Based on the theory of psychosomatic inhibition, we hypothesized that subjects who abstained from disclosing emotional material on a laboratory task would have poorer control of latent Epstein-Barr virus (as evidenced by high titers for the viral capsid antigen), and similarly, those subjects with psychometrically derived repressive interpersonal styles would show the highest Epstein-Barr viral capsid antigen titers (EBV-VCA). Eighty first-year undergraduates completed a personality inventory and were asked to write an essay/letter for 30 minutes about a stressful event that had happened in their life. Blood was collected from each subject immediately after writing. Essays were scored for degree of emotional disclosure according to the ratio of emotional-to-total words used. Degree of disclosure was found to be associated with impaired control of latent EBV (high antibody titers to the EBV-VCA) controlling for medication use, recent sleep loss, physical activity, lean body mass, caloric intake, and alcohol and recreational drug use. Further, individual differences in interpersonal style (characterized by emotional suppression) related to this immunologic marker in a similar fashion, and these two factors interacted in determining EBV-VCA titers. That is, Repressors who were either high or low disclosers had high levels of antibody titer to EBV-VCA, whereas only those Sensitizers who did not disclose had high antibody titers to EBV-VCA. In addition to supporting the hypothesis that emotional repression is associated with some aspects of host-virus interaction, the present findings highlight the importance of obtaining behavioral and psychometric assessments in psychoimmunologic investigations of this abstract affective construct (i.e., repression).     

Demonstration of URR-duplication variants of human papillomavirus type 6 in paraffin-embedded tissue sections of one condyloma acuminatum and one buschke-loewenstein tumour

Human papillomavirus type 6 (HPV 6) induces condylomata acuminata and laryngeal papillomas. Occasionally, HPV 6 may also be found in low-grade verrucous carcinomas. In some tumours, genetic analysis revealed the presence of HPV 6 variants with rearrangements, mostly DNA duplications, within the upstream regulatory region (URR). In this study, we analysed 98 formalin-fixed, paraffin-embedded condylomata acuminata obtained from 54 patients for the presence of URR-duplication variants of HPV 6. HPV 6 DNA could be amplified by polymerase chain reaction (PCR) from 40 samples. One condyloma acuminatum contained a HPV 6 genome with rearranged URR. Analysis using restriction enzyme cleavage suggested a DNA duplication within the URR of approximately 200 bp, spanning the Hpa II site at nt 7863 and the Dde I site at nt 7843 but not involving the Rsa I site at nt 7633. In addition, we analysed the distribution of the already characterized URR-duplication variant HPV 6_ACIB within different paraffin-embedded tissue sections of a Buschke-Loewenstein tumour. No correlation could be demonstrated between the presence of the rearranged genome and malignant histological changes. This result and the demonstration of an URR-duplication variant in a typical condyloma acuminatum suggest that duplications within the URR of HPV 6 are not directly related to malignant progression of HPV 6-induced tumours.     

HepG2细胞内HPVDNA物理状态及表达L1蛋白

目的了解人肝癌细胞系Hep G2细胞内人乳头瘤病毒(HPV)基因组的物理状态,胞质内包涵体物质的性质以及晚期衣壳蛋白1(L1)表达。方法用PCR对细胞内HPV18型E2和E6基因进行扩增,判断HPV18基因组的物理状态;用ELISA、光镜和电镜的免疫组化、Western blot等方法,以多价HPV L1小鼠单克隆抗体做探针,检测Hep G2细胞内L1蛋白表达;用反转录PCR检测细胞内L1 mRNA表达。结果 Hep G2细胞内HPV DNA基因组呈整合状态;细胞裂解液中有HPV L1蛋白存在;细胞呈HPV L1阳性反应;胞质内包涵体样物质,由均匀的颗粒样物质组成,可以为胶体金标记的HPV L1抗体所标识。Hep G2细胞裂解液中有HPV L1蛋白,在56 ku区出现与He La细胞一样的L1特异阳性条带。反转录PCR检测显示Hep G2细胞内有L1 mRNA存在。结论 Hep G2是HPV18阳性细胞,细胞内HPV DNA基因组呈整合状态。细胞内包涵体样物质为HPV18 L1蛋白,Hep G2细胞可以表达L1。     

机体对HPV L1免疫应答的研究进展

HPV L1相关的免疫应答是天然免疫和适应性免疫协同作用的结果,目前相关的研究已经渗入了免疫应答的全过程.天然免疫参与HPV L1诱导的免疫应答,但人们对它的认识仍然很有限.适应性免疫研究的进展主要体现在3方面①识别阶段.HPV的相关抗原主要由DC和LC摄取、加工、处理,经胞质溶胶、交叉提呈两种提呈途径提呈给CD8+细胞并使其致敏.②增殖分化阶段.Th1和Th2细胞都有出现,表明HPV L1不仅介导体液免疫,也介导细胞免疫.③效应阶段.L1介导产生的IgG、IgA、IgM类抗体都有其特殊性.