Paola Di Bonito

Survivin as a Marker of Cervical Intraepithelial Neoplasia and High-Risk Human Papillomavirus and a Predictor of Virus Clearance and Prognosis in Cervical Cancer

We analyzed survivin as a marker of cervical intraepithelial neoplasia (CIN) and high-risk human papillomavirus (HR-HPV) and a predictor of HPV clearance and disease outcome in cervical cancer in 302 samples (squamous cell carcinomas [SCCs], 150; CIN lesions, 152) by immunohistochemical staining with survivin antibody and HPV testing using polymerase chain reaction. HR-HPV types were associated closely with CIN and SCC. There was a significant linear relationship between grade and intensity of survivin expression (P = .0001). Survivin overexpression also was associated strongly with HR-HPV type (P = .0001). Multivariate regression analysis revealed survivin and p16(INK4a) as equally strong independent predictors of HR-HPV. Deregulated survivin expression did not predict clearance or persistence of HR-HPV after treatment of CIN or survival in cervical cancer in univariate (P = .417) or multivariate analysis. After adjustment for HR-HPV, stage, age, and tumor grade in the Cox regression model, only stage (P = .0001) and age (P = .0001) remained independent prognostic predictors. Survivin seems to be an early marker of cervical carcinogenesis. Up-regulated survivin expression was an independent predictor of HR-HPV in cervical lesions, most plausibly explained by its normal transcriptional repression by wild-type p53 being eliminated by HR-HPV E6 oncoprotein.

Anti-tumor CD8+ T cell immunity elicited by HIV-1-based virus-like particles incorporating HPV-16 E7 protein.

Here we report a novel strategy for the induction of CD8(+) T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef(mut)) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLP-associated Nef(mut) is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef(mut) led to an anti-E7 CD8(+) T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freunds adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef(mut)-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef(mut) VLPs a promising candidate for new vaccine strategies focused on the induction of CD8(+) T cell immunity.

Serum antibody response to Human papillomavirus (HPV) infections detected by a novel ELISA technique based on denatured recombinant HPV16 L1, L2, E4, E6 and E7 proteins

BackgroundHuman papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also involved in the development of other tumours (skin, head and neck). Serological survey of the HPV infections is important to better elucidate their natural history and to disclose antigen determinants useful for vaccine development. At present, the analysis of the HPV-specific antibodies has not diagnostic value for the viral infections, and new approaches are needed to correlate the antibody response to the disease outcome. The aim of this study is to develop a novel ELISA, based on five denatured recombinant HPV16 proteins, to be used for detection HPV-specific antibodies.MethodsThe HPV16 L1, L2, E4, E6 and E7 genes were cloned in a prokaryotic expression vector and expressed as histidine-tagged proteins. These proteins, in a denatured form, were used in ELISA as coating antigens. Human sera were collected from women with abnormal PAP smear enrolled during an ongoing multicenter HPV-PathogenISS study in Italy, assessing the HPV-related pathogenetic mechanisms of progression of cervical cancer precursor lesions. Negative human sera were collected from patients affected by other infectious agents. All the HPV-positive sera were also subjected to an avidity test to assess the binding strength in the antigen-antibody complexes.ResultsMost of the sera showed a positive reactivity to the denatured HPV16 proteins: 82% of the sera from HPV16 infected women and 89% of the sera from women infected by other HPV genotypes recognised at least one of the HPV16 proteins. The percentages of samples showing reactivity to L1, L2 and E7 were similar, but only a few serum samples reacted to E6 and E4. Most sera bound the antigens with medium and high avidity index, suggesting specific antigen-antibody reactions.ConclusionThis novel ELISA, based on multiple denatured HPV16 antigens, is able to detect antibodies in women infected by HPV16 and it is not genotype-specific, as it detects antibodies also in women infected by other genital HPVs. The assay is easy to perform and has low cost, making it suitable for monitoring the natural history of HPV infections as well as for detecting pre-existing HPV antibodies in women who receive VLP-based HPV vaccination.

Mucosal and cutaneous human papillomaviruses detected in raw sewages.

Epitheliotropic viruses can find their way into sewage. The aim of the present study was to investigate the occurrence, distribution, and genetic diversity of Human Papillomaviruses (HPVs) in urban wastewaters. Sewage samples were collected from treatment plants distributed throughout Italy. The DNA extracted from these samples was analyzed by PCR using five PV-specific sets of primers targeting the L1 (GP5/GP6, MY09/MY11, FAP59/64, SKF/SKR) and E1 regions (PM-A/PM-B), according to the protocols previously validated for the detection of mucosal and cutaneous HPV genotypes. PCR products underwent sequencing analysis and the sequences were aligned to reference genomes from the Papillomavirus Episteme database. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. A broad spectrum of sequences related to mucosal and cutaneous HPV types was detected in 81% of the sewage samples analyzed. Surprisingly, sequences related to the anogenital HPV6 and 11 were detected in 19% of the samples, and sequences related to the “high risk” oncogenic HPV16 were identified in two samples. Sequences related to HPV9, HPV20, HPV25, HPV76, HPV80, HPV104, HPV110, HPV111, HPV120 and HPV145 beta Papillomaviruses were detected in 76% of the samples. In addition, similarity searches and phylogenetic analysis of some sequences suggest that they could belong to putative new genotypes of the beta genus. In this study, for the first time, the presence of HPV viruses strongly related to human cancer is reported in sewage samples. Our data increases the knowledge of HPV genomic diversity and suggests that virological analysis of urban sewage can provide key information useful in supporting epidemiological studies.

Predicting high-risk human papillomavirus infection, progression of cervical intraepithelial neoplasia, and prognosis of cervical cancer with a panel of 13 biomarkers tested in multivariate modeling.

Summary: Comprehensive multivariate models were used to disclose whether any of our previously analyzed 13 markers would be independent predictors of intermediate end point markers in cervical carcinogenesis. The expression of the following biomarkers, E-cadherin, extracellular signal-regulated kinase 1, 67-kd laminin receptor (LR67), matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 2, nuclear factor-&kgr;B, nm23-H1, p16INK4A, proliferating cell nuclear antigen, survivin, human telomerase reverse transcriptase, topoisomerase 2&agr;, and vascular endothelial growth factor (VEGF) C in 150 cervical cancer (CC) and 152 cervical intraepithelial neoplasia (CIN) lesions were determined immunohistochemically. Multivariate models were constructed to test predictive power of the markers for 3 outcomes: (1) high-grade CIN, (2) high-risk human papillomavirus (HR-HPV), and (3) CC survival. Performance indicators were calculated and compared by the areas under receiver operating characteristic (ROC) curve. Three marker panels were identified consisting of 5 independent predictors of CIN2 (E-cadherin, extracellular signal-regulated kinase 1, LR67, topoisomerase 2&agr;, and VEGF-C), 3 predictors of HR-HPV (survivin, p16INK4a, and human telomerase reverse transcriptase), and 2 predictors of CC survival (nm23-H1 and tissue inhibitor of metalloproteinase 2). In predicting CIN2, the best balance between sensitivity (SE) and specificity (SP) was obtained by combining the 2 most powerful predictors in panel 1 (VEGF-C and LR67), giving the area under ROC curve, 0.897 (95% confidence interval [CI], 0.847-0.947); odds ratio, 86.27 (95% CI, 19.71-377.47); SE, 86.0%; SP, 93.3%; positive predictive value (PPV), 99.1%; and negative predictive value (NPV), 43.1%. In a hypothetical screening setting (10,000 women; CIN2 prevalence, 1%), this marker combination should theoretically detect CIN2 with 86.0% SE, 100% SP, 99.1% PPV, and 99.6% NPV, area under ROC curve of 0.930 (95% CI, 0.909-0.951), and odds ratio, 29998.0 (95% CI, 7,879.0-37,338.0). Combining 2 markers (LR67 and VEGF-C) enables accurate detection of high-grade CIN in a clinical setting. However, testing the performance of this marker combination in a screening setting necessitates their analysis in cytological samples.

Seroprevalence of Toscana virus in blood donors, France, 2007.

To the Editor: Toscana virus (TOSV) is an arthropod-borne RNA virus (family Bunyaviridae and genus Phlebovirus) transmitted by sandflies in Mediterranean countries. TOSV causes acute meningitis and meningoencephalitis in patients. In France, cases of TOSV infections involving resident populations and cases imported by tourists traveling in TOSV-endemic countries have been reported (1,2); the virus has also been isolated from local wild-caught sandflies (1). The fact that TOSV has been isolated from human blood on several occasions (2) suggests a potential risk exists for transmitting the virus through blood transfusion or organ transplantation. We investigated the presence of TOSV antibodies in a sample of the healthy population, blood donors from southeastern France. We tested plasma collected from 729 blood donors in 7 French territorial divisions during the summer of 2007. Plasma donors were analyzed according to their address of residence in each territorial division. Information related to these donors is reported in the Table. Table Prevalence of antibodies against Toscana virus in blood donors, France, 2007* Presence of immunoglobulin (Ig) G and IgM against TOSV was investigated by using a commercial enzyme immunoassay kit (EIA Enzywell Toscana virus IgG and IgM; DIESSE Diagnostica Senese S.p.A., Siena, Italy) developed by using the recombinant nucleocapsid (N) protein of TOSV. This serologic test was validated in a previous study that revealed high specificity and sensitivity (3). Our results showed that 84 (11.5%) of 729 plasma samples were positive for IgG against TOSV N protein. Twenty-four (3.3%) plasma samples were positive for IgM, and 5 (0.7%) were positive for IgG and IgM (Table). To confirm the ELISA results, IgG-positive samples were further subjected to Western blot (WB) analysis by using TOSV (isolate H/IMTSSA [2])–infected cell lysate (4). In 233 (32%) of samples, we detected a protein of molecular mass compatible with that of the N protein. A previously reported antibody-positive control was used to validate the WB assay (5). Our WB analysis showed a reduced sensitivity when compared with results of ELISA. After chemical/heat treatment of the protein samples, WB will only detect the linear epitopes on the N protein, while ELISA detects both linear and conformational epitopes. Furthermore, a less recent exposure of the blood donor population to the virus would have resulted in weaker N protein detection by WB as a consequence of a lower antibody titer. However, we cannot exclude some aspecific cross-reactivity as a consequence of well-conserved N protein sequence among the genus. Finally, to detect TOSV RNA, we processed IgM-positive plasma samples by reverse transcription–PCR (6). The finding of IgM is an indication of a recent exposure to the virus and hence a possible presence in blood. Our PCR did not detect any viral RNA in the samples. Such negative results could indicate either cleared viremia or a low viral load, below the sensitivity limit of the test. Serologic information obtained in our study confirms the circulation of TOSV in southeastern France. Factors such as commercial exchange and movement of humans, animals, and arthropods between France and Italy may explain the highest prevalence observed (18.8%) in the Alpes Maritimes territorial district, which borders Italy. Our results regarding this area appear of the same order of magnitude as those reported in the general Italian population (>20%) (1). Geographic and climatic conditions (e.g., temperature, humidity), factors that affect vector distribution and abundance (7), could explain the lower prevalence found in the mountainous districts (collectively ≈400–2,000 meters in elevation). The lower temperatures in these districts may also affect the ability of vectors to efficiently transmit the virus in the field (8). TOSV prevalence in Corsica, an island in the Mediterranean Sea, was unexpectedly high. In this region, ≈8.7% (10 donors of 115) of the population sampled showed an IgG- or IgM-positive response. In the other districts, the IgM seroprevalence did not exceed 4.4%. The vector that transmits TOSV is known to be present in this area (7), and TOSV infections have been reported on nearby Sardinia (9). The elevated IgM titer in the population in Corsica could indicate 1) recent virus contacts; 2) recent infections with a new TOSV strain circulating in Corsica; or 3) presence of related phleboviruses that are inducing cross-reactivity in the N protein–based IgM ELISA. Our results demonstrate that 14.1% (IgG and IgM) of the healthy population (blood donors) in France living on the Mediterranean border have been in contact with TOSV and show asymptomatic or mild, unidentified symptoms, as it is the case for many other arbovirus infections (10). Such findings raise concerns about the risks of virus transmission to virus-naive persons by blood transfusions and organ transplants. Further investigation is needed to better assess how widespread TOSV is in populations. For example, a donor–recipient investigation might confirm virus transmission by blood transfusion, and studies related to the behavior of sandfly vectors, virus biology, and mammalian reservoir hosts could help define populations at higher risk for exposure.

HPV-E7 Delivered by Engineered Exosomes Elicits a Protective CD8+ T Cell-Mediated Immune Response

We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

In vivo antitumor effect of an intracellular single-chain antibody fragment against the E7 oncoprotein of human papillomavirus 16

Human papillomavirus (HPV)‐associated tumors still represent an urgent problem of public health in spite of the efficacy of the prophylactic HPV vaccines. Specific antibodies in single‐chain format expressed as intracellular antibodies (intrabodies) are valid tools to counteract the activity of target proteins. We previously showed that the M2SD intrabody, specific for the E7 oncoprotein of HPV16 and expressed in the endoplasmic reticulum of the HPV16‐positive SiHa cells, was able to inhibit cell proliferation. Here, we showed by confocal microscopy that M2SD and E7 colocalize in the endoplasmic reticulum of SiHa cells, suggesting that the E7 delocalization mediated by M2SD could account for the anti‐proliferative activity of the intrabody. We then tested the M2SD antitumor activity in two mouse models for HPV tumors based respectively on TC‐1 and C3 cells. The M2SD intrabody was delivered by retroviral vector to tumor cells before cell injection into C57BL/6 mice. In both models, a marked delay of tumor onset with respect to the controls was observed in all the mice injected with the M2SD‐expressing tumor cells and, importantly, a significant percentage of mice remained tumor‐free permanently. This is the first in vivo demonstration of the antitumor activity of an intrabody directed towards an HPV oncoprotein. We consider that these results could contribute to the development of new therapeutic molecules based on antibodies in single‐chain format, to be employed against the HPV‐associated lesions even in combination with other drugs.

Recombinant HPV16 E7 assembled into particles induces an immune response and specific tumour protection administered without adjuvant in an animal model.

BackgroundThe HPV16 E7 protein is both a tumour-specific and a tumour-rejection antigen, the ideal target for developing therapeutic vaccines for the treatment of HPV16-associated cancer and its precursor lesions. E7, which plays a key role in virus-associated carcinogenesis, contains 98 amino acids and has two finger-type structures which bind a Zn++ ion. The ability of an Escherichia coli-produced E7-preparation, assembled into particles, to induce protective immunity against a HPV16-related tumour in the TC-1-C57BL/6 mouse tumour model, was evaluated.MethodsE7 was expressed in E. coli, purified via a one-step denaturing protocol and prepared as a soluble suspension state after dialysis in native buffer. The presence in the E7 preparation of particulate forms was analysed by non-reducing SDS-PAGE and negative staining electron microscopy (EM). The Zn++ ion content was analysed by mass-spectrometry. Ten μg of protein per mouse was administered to groups of animals, once, twice or three times without adjuvant. The E7-specific humoral response was monitored in mice sera using an E7-based ELISA while the cell-mediated immune response was analysed in mice splenocytes with lymphoproliferation and IFN-γ ELISPOT assays. The E7 immunized mice were challenged with TC-1 tumour cells and the tumour growth monitored for two months.ResultsIn western blot analysis E7 appears in multimers and high molecular mass oligomers. The EM micrographs show the protein dispersed as aggregates of different shape and size. The protein appears clustered in micro-, nano-aggregates, and structured particles. Mice immunised with this protein preparation show a significant E7-specific humoral and cell-mediated immune response of mixed Th1/Th2 type. The mice are fully protected from the tumour growth after vaccination with three E7-doses of 10 μg without any added adjuvant.ConclusionsThis report shows that a particulate form of HPV16 E7 is able to induce, without adjuvant, an E7-specific tumour protection in C57BL/6 mice. The protective immunity is sustained by both humoral and cell-mediated immune responses. The E. coli-derived HPV16 E7 assembled in vitro into micro- and nanoparticles represents not only a good substrate for antigen-presenting cell uptake and processing, but also a cost-effective means for the production of a new generation of HPV subunit vaccines.

Intracellular anti-E7 human antibodies in single-chain format inhibit proliferation of HPV16-positive cervical carcinoma cells

The E7 tumor antigen of human papillomavirus type 16 (HPV16) is a validated target for immunodiagnosis and immunotherapy of HPV‐associated cervical cancer. Anti‐HPV16 E7 antibodies in scFv format were isolated from a human antibody phage display library and characterized. With the aim of interfering with the oncogenic activity of E7 protein, the most reactive of the selected antibody fragments was expressed by eukaryotic vectors in different compartments of the HPV16‐positive cervical carcinoma SiHa cell line. The intracellular antibodies (intrabodies) were tested for their ability of inhibiting cell proliferation. A significant inhibition was obtained targeting the intrabodies to the nuclear and secretory compartments whereas no significant effect was observed in case of cytoplasmic localization. Inhibition was highly specific as no antiproliferative effect was obtained either with the E7‐specific intrabodies in HPV‐negative cells nor with irrelevant intrabodies in SiHa cells.