Frequency of antibodies against E4 and E7 from human papillomavirus type 16 in Mexican soldiers

Summary. The high prevalence of HPV in men’s genitalia and the low frequency of virus-associated lesions gave rise to questions on the influence of infection-site on the HPV antibody profile. In a cross-sectional study, HPV infection in penis and urethra, and serum antibodies against HPV-16 E4 and E7 proteins were evaluated in 288 Mexican soldiers. The results showed that HPV prevalence was 31% (51% in penis, 11% in urethra and 38% in both sites), while 47% were multiple infections. Overall, seroprevalence was 13% for anti-E4 antibodies and 6% for anti-E7. However, the highest prevalence of anti-E4 antibodies was observed in men with HPV infection in urethra (30%), while for E7 antibodies, the highest prevalence (10%) was found in men who tested positive for HPV in penis. The prevalence of IgG and IgA anti-E4 was related to HPV-16 urethral infection, while detection of HPV-16 in penis was related to IgG anti-E7 prevalence. In conclusion, the high-risk sexual behavior observed in this population might be responsible for high HPV prevalence and multiple infections. However, the seroprevalence of E4 and E7 was similar to that observed in healthy Mexican women. These results suggest that the humoral immune response against HPV infection in men differs, depending on the site of infection.     

E6 and E7 oncoproteins from human papillomavirus type 16 induce activation of human transforming growth factor beta1 promoter throughout Sp1 recognition sequence

Human Papillomavirus (HPV) infection is the main etiologic agent of cervical cancer and HPV E6 and E7 oncogenes trans-regulate many cellular genes. An association between TGF-beta1 gene expression and cervical cancer development has been suggested; however, the mechanisms by which HPV influences TGF-beta1 expression remain unclear. In the present study we analyzed the mechanism through which HPV-16 E6 and E7 oncoproteins regulate the TGF-beta1 promoter in cervical tumor cells. Our results showed that E6 and E7 increased TGF-beta1 promoter activity. Furthermore, we identified a specific DNA sequence motif in the TGF-beta1 core promoter that is responsible for trans-activation and that corresponds to the Sp1e-binding site associated with HPV-16 E6 and E7 oncoproteins. Mutational analysis showed that the Sp1e recognition site abolished the trans-activation caused by E6 and E7. These results suggest a physical interaction and functional cooperation between viral oncoproteins and cellular regulatory elements of the TGF-beta1 promoter, and may explain the contribution of HPV-16 to TGF-beta1 gene expression in cervical cancer.     

Purification and characterisation of the E7 oncoproteins of the high-risk human papillomavirus types 16 and 18

E7 proteins are major oncoproteins of human papillomaviruses (HPVs) which play a key role in virus-associated cervical carcinogenesis. The E7 oncoprotein of HPV-16 has been shown to interact with a variety of cellular target proteins and these interactions are considered essential for the transforming properties of this oncoprotein. Several additional HPV types associated etiologically to cervical cancer have been described, the second most common being HPV-18. Less is known about the biochemical functions and interactions of HPV-18 E7. As a first step to determine biochemical properties common to the E7 proteins of the high-risk HPV types 16 and 18 these E7 proteins were expressed in bacteria and purified to homogeneity. Purified E7 proteins were used to investigate the in vitro interaction with the pocket protein p107 and insulin-like growth factor-binding protein-3 (IGFBP-3) that are known to interact with the amino-terminal and the carboxyl-terminal part of IGFBP-3, respectively. Both purified E7 proteins interacted strongly with p107 and, as demonstrated here for the first time, HPV-18 E7 was capable of binding to IGFBP-3, albeit to a lesser extent than HPV-16 E7. These findings suggest that the purified recombinant E7 proteins retain, at least in part, their biochemical activities.     

Viral recombinant vaccines to the E6 and E7 antigens of HPV-16

Most cancerous lesions of the uterine cervix are linked to persistent infections with human papillomaviruses (HPV), most notably HPV-16 or -18. Vaccine-induced immune responses to the HPV early antigens E6 and E7, which contribute to cell transformation and are thus expressed in these cervical cancers, could potentially eradicate malignant cells. We generated recombinant vaccines based on E1-deleted adenovirus human strain 5 or on vaccinia virus strain Copenhagen expressing either the E6 or E7 oncoproteins of HPV-16. The different vaccines were compared in two experimental mouse tumor models employing Balb/c or C57Bl/6 mice. Data presented here demonstrate that depending on the model either CD4(+) or CD8(+) T cells provide protection to tumor cell challenge, resulting in striking differences in the efficacy of the four vaccines under investigation.     

Expression of human papillomavirus type 16 E7 protein by recombinant baculovirus and use for the detection of E7 antibodies in sera from cervical carcinoma patients

Although the presence of serum antibodies against the human papillomavirus type 16 (HPV-16) E7 protein has been linked with cervical cancer, currently available assays detect antibodies in only ca. 40% of carcinoma patients. The dependence of these serological assays on synthetic target antigens which present only linear epitopes may be a limiting factor. In order to produce a more realistic target antigen for use in serological assays, we have expressed the HPV-16 E7 protein in insect cells using a recombinant baculovirus vector. Two major E7 forms of ca. 18kDa and 16kDa were produced and characterised. The 16kDa component was shown to be truncated at the N-terminus. A radioimmunoprecipitation assay was developed for the detection of anti-E7 antibodies in human sera. This assay showed a marked increase in detection rate compared with a western blotting method based on bacterially derived E7 fusion proteins. © 1993 Wiley-Liss, Inc.     

Phage display for site-specific immunization and characterization of high-risk human papillomavirus specific E7 monoclonal antibodies

The paper describes a method to use filamentous phage to display specific regions of proteins for immunization in order to direct the immune response towards a pre-defined region of the protein. The method called site-specific immunization (SSI) was evaluated using the E7 protein of oncogenic (high-risk) human papillomavirus (HPV) type 16 as a model system. This protein consists of sequence blocks also present in other viral and cellular proteins and in the corresponding protein of low-risk HPVs. A fragment of the HPV16 E7 oncoprotein specific for a group of high-risk viruses was identified by sequence comparison and displayed on filamentous phages in fusion with the major phage coat protein VIII. The recombinant phages triggered an immune response in mice against the full-length HPV16 E7 protein. Fusion of B-lymphocytes from the immunized animals with myeloma cells resulted in three hybridomas producing monoclonal antibodies (MAbs) with reactivity against the endogenous E7 protein. The specificity of the MAbs for the HPV16 E7 protein in cancer cell lines was confirmed by Western blot analyses and immunocytochemistry. The epitope of each MAb was roughly mapped by determining the reactivity against overlapping E7 fragments displayed on phage particles. The mimotopes of the MAbs were further determined by biopanning against a randomized peptide library displayed on phage and found to be unique for a sub-set of high-risk HPV E7 proteins. The combination of different phage display techniques for immunization and epitope mapping was efficient for generation and characterization of highly specific MAbs.     

The stability of the human papillomavirus E6 oncoprotein is E6AP dependent

Human papillomavirus (HPV) E6 oncoproteins target numerous cellular proteins for ubiquitin-mediated degradation. In the case of p53 this is mediated by the E6AP ubiquitin ligase. However, there are conflicting reports concerning how central E6AP is to the global function of the HPV-16 and HPV-18 E6 oncoproteins. To investigate this further we have analysed the effects of E6AP removal upon the stability of endogenously expressed E6 protein. We show that when E6AP is silenced in HPV-positive cells, E6 protein levels are dramatically decreased in a proteasome-dependent manner. Further, we show that when E6AP is depleted in HeLa cells, E6 has a greatly decreased half-life. In addition, overexpression of E6AP stabilises ectopically expressed HPV-16 and HPV-18 E6 in a manner that is independent of its ubiquitin ligase activity. These results demonstrate that the stability of HPV E6 is critically dependent upon the presence of E6AP.     

Antibodies to HPV-16 E6 and E7 proteins as markers for HPV-16-associated invasive cervical cancer.

Transforming proteins E6 and E7 of human papillomaviruses (HPVs) are consistently expressed in HPV-associated cervical cancers. In ELISA with four HPV-16 E6-E7 peptides, patients with HPV-16-associated invasive cervical cancer (group 1) had a greater seroreactivity than all other groups, which included patients with HPV-16-associated cervical intraepithelial neoplasia, invasive cervical cancer patients without HPVs, and unaffected controls. A larger proportion of group 1 sera, as compared to sera of all other groups, was reactive with at least one peptide (49% vs 17-27%), and with two or more peptides (22% vs 0-6%). A clear difference between group 1 and all other groups was also found for high ELISA absorbance values to at least one peptide (22% vs 0-8%). This high seroreactivity of group 1 sera was confirmed by a radioimmunoprecipitation assay with in vitro transcribed and translated HPV-16 E7 protein. Sera from 50% of group 1 but only 3% of controls were reactive in this test. Antibodies to HPV-16 E6 and E7 proteins appear to be virus-specific and disease state-specific markers of HPV-associated cervical cancer.     

Independent association of antibodies against human papillomavirus type 16 E1/E4 and E7 proteins with cervical cancer.

The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an E1/E4 fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac-E1/E4 and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti-E1/E4 antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti-E1/E4 and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.     

The E6E7 oncoproteins of cutaneous human papillomavirus type 38 interfere with the interferon pathway

Non-melanoma skin cancer is the most frequent malignancy in Caucasian populations. Evidence suggests the involvement of cutaneous Human Papillomavirus (HPV) of the genus beta (beta) in this disease. The ability of E6 and E7 of mucosal HPV to promote cellular transformation and inhibit immune response-related pathways plays a key role in cervical carcinogenesis. beta HPV-38 E6 and E7 display transforming activities in in vitro and in vivo models, but their impact on immune surveillance is unknown. Here we show that HPV-38 E6 and E7 affect the IFN-induced up-regulation of MHC class I. Expression of the two viral proteins in HaCaT keratinocytes led to a decrease of MHC I levels. This down-regulation is associated with a reduction of expression of MHC I heavy chain, of the peptide chaperone TAP and of the STAT-1 downstream effector IRF-1. The down-regulation of these proteins is ultimately due to the inhibition of STAT-1 expression. Analysis of cells expressing either HPV-38 E6 or E7 suggests that these effects are primarily the result of E6 expression, although a contribution by E7 cannot be excluded. We conclude that HPV-38 encodes oncoproteins that potentially contribute to the evasion of host immune surveillance.     

Human papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti-E7 prevalence in cervical cancer patients.

Antibody-reactive regions on the human papillomavirus type 18 (HPV-18) E6 and E7 proteins were identified with rabbit polyclonal anti-fusion protein sera by screening of an fd phage expression library containing subgenomic HPV-18 DNA fragments and by testing of overlapping decapeptides representing the E6 and E7 open reading frames. Peptides comprising the delineated regions (designated E6/1 to E6/4 and E7/1) were synthesized and used in an enzyme-linked immunosorbent assay (ELISA) to detect anti-HPV-18 antibodies in human sera. A total of 232 human serum samples (identical numbers of cervical cancer patients and age-matched controls) collected in Tanzania were tested. Similar prevalences (between 0.8 and 4.3%) of antibodies recognizing the different E6 peptides were found in the sera from tumor patients and controls. With a synthetic 28-mer peptide (designated pepE701) comprising the E7/1 region, a significant difference was found: 10 of 116 tumor serum samples but 0 of 116 control serum samples showed a specific reaction (P less than 0.001). This observation confirms earlier results with HPV-16 E7 fusion proteins (I. Jochmus-Kudielka, A. Schneider, R. Braun, R. Kimmig, U. Koldovsky, K. E. Schneweis, K. Seedorf, and L. Gissmann, J. Natl. Cancer Inst. 81:1698-1704, 1989). A lower prevalence of anti-HPV-18 E7 antibodies was observed when 188 human serum samples collected in Germany from tumor patients and controls were tested (3 of 94 positive in the cancer group; 0 of 94 positive in the control group). The type specificity of anti-HPV-18 E7 antibodies was demonstrated when the HPV type found by Southern hybridization in the cervical cancer biopsies was compared with seroreactivity: 4 of 8 serum samples obtained from HPV-18 DNA-positive but 0 of 16 serum samples from HPV-18 DNA-negative tumor patients reacted in the HPV-18 E7 ELISA. In addition, HPV-18-positive sera failed to react in a peptide ELISA with the homologous HPV-16 E7 region (M. Müller, H. Gausepohl, G. de Martinoff, R. Frank, R. Brasseur, and L. Gissmann, J. Gen. Virol. 71:2709-2717, 1990) and vice versa.     

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.     

Identification in humans of HPV-16 E6 and E7 protein epitopes recognized by cytolytic T lymphocytes in association with HLA-B18 and determination of the HLA-B18-specific binding motif

Human papilloma virus type 16 (HPV-16) is the HPV most frequently associated with cervical carcinoma in humans. For the prevention or treatment of cervical carcinoma, the E6 and E7 oncoproteins appear to be good targets for vaccine-induced cytotoxic T lymphocytes (CTL). Lipopeptide vaccination is an efficient way of stimulating cellular responses. However, to synthesize effective lipopeptides, it is necessary to define which epitopes are immunogenic. In this study we first determined that peptide 80 - 88 of the E6 protein was recognized by CTL from a healthy donor in association with the HLA-B18 molecule. We then defined the HLA-B18 anchoring peptide motif by testing the binding of various short peptides with the HLA-B18 molecule and showed that it was related to the HLA-A1-specific peptide motif. Furthermore, in analyzing the potential E7 epitopes susceptible to associating with HLA-B18, we demonstrated that peptide E7 44 - 52 gave the strongest binding. It could also be recognized by CTL from peripheral blood mononuclear cells (PBMC) of the same healthy donor. Finally, with PBMC from a patient with a cervical intraepithelial neoplasia grade 3, we found CTL which recognized the E6 80 - 88 epitope. We have hence identified two peptides encoded by the E6 and E7 proteins which are presented by the HLA-B18 molecule and could be included in a vaccine against HPV-16.     

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.     

Follow-up of antibody responses to human papillomavirus type 16 E7 in patients treated for cervical carcinoma

A synthetic peptide comprising amino acids 6-35 of HPV-16 E7 was used in an ELISA to screen sera taken from 31 cervical carcinoma patients. Sera obtained before and during treatment, and in follow-up, were tested for the presence of antibodies to this peptide. Sixteen patients with negative pretreatment serum determination remained negative during treatment and follow-up. Of the 15 patients with positive pretreatment sera, 12 showed a decrease in anti-E7 6–35 antibody level during treatment. During follow-up an increase in anti-E76–35 antibody level was observed in 6 out of 7 patients with progressive or recurrent disease, whereas all patients who remained in complete remission showed stable or further decreasing antibody levels. During the course of disease of the 15 seropositive patients, serum anti-E76–35 antibody levels were compared with serum squamous cell carcinoma antigen (SCC-Ag) profiles, a clinically useful tumor marker in the management of cervical cancer patients. Similar patterns were observed in 10 out of 15 patients. The results of this study suggest that in a subset of cervical cancer patients, anti-E76–35 antibody response against HPV-16 E7 at least partially depends on the presence of viable tumor lesions, and that to some extent the anti-E7 profile reflects the course of disease. © 1995 Wiley-Liss, inc.