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.

Successful therapeutic vaccination with integrase defective lentiviral vector expressing nononcogenic human papillomavirus E7 protein.

Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.

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

BACKGROUND: The 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. METHODS: E7 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. RESULTS: In 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. CONCLUSIONS: This 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.

Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells.

BACKGROUND: "High risk" human papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins. METHODS: In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™ technology and Surface Plasmon Resonance. RESULTS: The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner. CONCLUSIONS: Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic approaches against the HPV-associated lesions.

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.

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-kappaB, nm23-H1, p16, proliferating cell nuclear antigen, survivin, human telomerase reverse transcriptase, topoisomerase 2alpha, 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 2alpha, and VEGF-C), 3 predictors of HR-HPV (survivin, p16, 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.

Characterization of antibodies in single-chain format against the E7 oncoprotein of the human papillomavirus type 16 and their improvement by mutagenesis.

BACKGROUND: Human papillomaviruses (HPV) are the etiological agents of cervical cancer. The viral E7 protein plays a crucial role in viral oncogenesis. Many strategies have been explored to block the E7 oncoprotein activity. The single-chain variable antibody fragments (scFvs) are valuable tools in cancer immunotherapy and can be used as "intracellular antibodies" to knock out specific protein functions. For both in vivo and in vitro employment, the scFv intrinsic solubility and stability are important to achieve long-lasting effects. Here we report the characterization in terms of reactivity, solubility and thermal stability of three anti-HPV16 E7 scFvs. We have also analysed the scFv43 sequence with the aim of improving stability and then activity of the antibody, previously shown to have antiproliferative activity when expressed in HPV16-positive cells. METHODS: The three anti-HPV16 E7 scFv 32, 43 51 were selected from the ETH-2 "phage-display" library. Thermal stability was evaluated with ELISA by determining the residual activity of each purified scFv against the recombinant HPV16 E7, after incubation in the presence of human seroalbumine for different time-intervals at different temperatures. Sequence analysis of the scFvs was performed with BLAST and CLUSTALL programs. The scFv43 aminoacid changes were reverted back to the consensus sequence from the immunoglobuline database by site-directed mutagenesis. ScFv solubility was evaluated with Western blotting by determining their relative amounts in the soluble and insoluble fractions of both prokaryotic and eukaryotic systems. RESULTS: ScFv51 was the most thermally stable scFv considered. Sequence analysis of the most reactive scFv43 has evidenced 2 amino acid changes possibly involved in molecule stability, in the VH and VL CDR3 regions respectively. By mutagenesis, two novel scFv43-derived scFvs were obtained, scFv43 M1 and M2. ScFv43 M2 showed to have improved thermal stability and solubility in comparison with the parental scFv43. CONCLUSION: The characterization of 5 specific anti-HPV16 E7 scFvs shows features important for their activity in vivo. ScFv43 M2 shows higher thermal stability with respect to the parental scFv43, and scFv51 shows high stability and solubility. These properties make the 2 scFvs the best candidates to be tested for anti-E7 activity in vivo.

Relationship of up-regulation of 67-kd laminin receptor to grade of cervical intraepithelial neoplasia and to high-risk HPV types and prognosis in cervical cancer.

OBJECTIVE: To evaluate the 67-kd laminin receptor (67LR) in cervical cancer and its molecular links to oncogenic HPV types. STUDY DESIGN: As part of the HPV-PathogenlSS Study, a series of 150 squamous cell carcinomas (SCCs) and 152 carcinoma in situ (CIN) lesions were examined using immunohistochemical staining for LR67 and tested for HPV using polymerase chain reaction (PCR) with 3 primer sets (MY09/11, GP5+/GP6+, SPF). Followup data were available for all SCC patients, and 67 CIN lesions had been monitored with serial PCR for HPV clearance/persistence after cone treatment. RESULTS: 67LR expression increased in parallel with increasing grade of CIN (p = 0. 0001), with the most dramatic up-regulation upon the transition from CIN 2 to CIN 3 and further to SCC. This increased expression was associated with CIN 3/cancer at OR 17.04 (95% CI 7.28-39.87). The seemingly significant association of 67LR with high-risk HPV (HR-HPV) detection (OR 2.20, 95% CI 1.27-3.80) was due to confounding by the histologic grade (Mantel-Haenszel common OR = 1.118, 95% CI 0.576-2.168). Using performance indicators, 67LR expression was of little value as a marker of HR-HPV type, and it did not predict clearance/persistence of HR-HPV after treatment of CIN. Similarly, 67LR expression was not an independent prognostic factor in cervical cancer. CONCLUSION: In cervical carcinogenesis, both integrin- and nonintegrin-type LRs (67LR) probably have functions complementary to each other, mediating transient early and stable adhesions, respectively. Up-regulated 67LR expression is significantly associated with progression from CIN 2 to CIN 3 as a marker of cell proliferation. 67LR is probably orchestrated by mechanisms independent of HR-HPV oncoproteins, which seem to be more closely associated with integrin-type laminin receptors.

Plant-derived human papillomavirus 16 E7 oncoprotein induces immune response and specific tumor protection.

Vaccine strategies for treatment of human papillomavirus-induced cervical cancer are based on either the recombinant E7 fusion oncoprotein or E7 CTL peptides. The therapeutic potential of the E7-based vaccine depends on the use of different adjuvants. In this study, we describe for the first time the expression of the human papillomavirus 16 E7 protein in Nicotiana benthamiana plant using a potato virus X-derived vector. C57BL/6 mice immunized with E7-containing crude foliar extracts developed both humoral and cell-mediated immune responses and were protected from tumor development after challenge with the E7-expressing C3 tumoral cell line. Our data support the possibility of producing a cost-effective anticancer vaccine in plant with intrinsic adjuvant-like properties.

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.

Activation of the ERK/MAP kinase pathway in cervical intraepithelial neoplasia is related to grade of the lesion but not to high-risk human papillomavirus, virus clearance, or prognosis in cervical cancer.

We subjected 302 archival samples (150 squamous cell carcinomas [SCCs] and 152 cervical intraepithelial neoplasia [CIN] lesions) to immunohistochemical staining with extracellular signal-regulated kinase-1 (ERK1) antibody and human papillomavirus (HPV) testing with 3 primer sets. Follow-up data were available for all SCC cases and 67 CIN cases. High-risk (HR) HPV types were associated with CIN (odds ratio [OR], 19.12; 95% confidence interval [CI], 2.31-157.81) and SCC (OR, 27.25; 95% CI, 3.28226.09). There was a significant linear relationship between lesion grade and ERK1 staining intensity (P = .0001). ERK1 staining was a 100% specific indicator of CIN, with a 100% positive predictive value, but a poor predictor of HR HPV. ERK1 expression did not predict clearance or persistence of HR HPV after CIN treatment. ERK1 staining did not significantly predict survival in cervical cancer in univariate (P = .915) 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 = .002) remained independent prognostic factors. ERK1 expression seems to be an early marker of cervical carcinogenesis. ERK1 overexpression is not a specific marker of HR-HPV in CIN and cervical cancer, nor does it predict virus clearance after CIN treatment or disease outcome in cervical cancer.

Human papillomavirus vaccines in plants.

Human papillomaviruses are the etiological agents of cervical cancer, one of the two most prevalent cancers in women in developing countries. Currently available prophylactic vaccines are based on the L1 major capsid protein, which forms virus-like particles when expressed in yeast and insect cell lines. Despite their recognized efficacy, there are significant shortcomings: the vaccines are expensive, include only two oncogenic virus types, are delivered via intramuscular injection and require a cold chain. Plant expression systems may provide ways of overcoming some of these problems, in particular the expense. In this article, we report recent promising advances in the production of prophylactic and therapeutic vaccines against human papillomavirus by expression of the relevant antigens in plants, and discuss future prospects for the use of such vaccines.

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 Freund's 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.

Guidelines of the Italian Society for Virology on HPV testing and vaccination for cervical cancer prevention.

OBJECTIVE: To provide guidelines for health-care providers on strategies for cervical cancer prevention based on HPV testing and anti-HPV vaccination. OUTCOMES: Overall efficacy of different preventive strategies, assessing reduction in the incidence of invasive cervical cancer and precancerous lesions. EVIDENCE: Medline and the Cochrane Database were searched for articles in English on subjects related to HPVs, HPV diagnosis, HPV anogenital lesions, cervical cancer, HPV testing, and HPV vaccines, in order to elaborate an up-dated document. Relevant Italian Government publications and position papers from appropriate health and family planning organizations were also reviewed. VALUES: The quality of the evidence and ranking of recommendations for practice were rated using criteria defined by SIV, which were adapted from the Canadian Task Force on Preventive Health Care.

Immunization with Toscana virus N-Gc proteins protects mice against virus challenge.

Toscana virus (TOSV) is an emerging virus, circulating in the Mediterranean area, that is responsible for aseptic meningitis, meningoencephalitis, and encephalitis. The development of a vaccine that could provide complete protection from TOSV infection is needed. In this study we investigated the capacity of TOSV structural proteins, nucleocapsid protein N and the two Gc and Gn glycoproteins, produced as recombinant proteins, in an animal model. In particular, we investigated their role in inducing specific and protective immune responses against virus infection. Mice were immunized intraperitoneally using TOSV antigens singly or in combination. The results show that only the N-Gc combination was able to protect 100% of animals from a lethal challenge with a neurovirulent strain of TOSV. This potential vaccine induces high serum antibody titres with neutralizing activity and it is safe for animals. Moreover, immunization induces a virus specific cell-mediated immune response, in particular a CD8+ T cell response associated with a marked expression of interferon gamma. These results indicate that the N+Gc viral antigen combination could be useful for future development of a vaccine controlling the spread of this emerging virus that could pose a new threat for humans.

Clinical and epidemiological correlates of antibody response to human papillomaviruses (HPVs) as measured by a novel ELISA based on denatured recombinant HPV16 late (L) and early (E) antigens.

BACKGROUND: At present, seroreactivity is not a valuable parameter for diagnosis of Human Papillomavirus (HPV) infection but, it is potentially valuable as marker of viral exposure in elucidating the natural history of this infection. More data are needed to asses the clinical relevance of serological response to HPV. OBJECTIVES: The objective was to assess the clinical and epidemiological correlates of HPV-seroreactivity in a cohort of HIV-negative and HIV-positive women. METHODS: Seroreactivity of 96 women, evaluated in an ELISA test based on denatured HPV16 late (L) and early (E) antigens, was correlated with their clinical and epidemiological data previously collected for a multi-centre Italian study, HPV-PathogenISS study. RESULTS: No significant correlation was found between HPV DNA detection and seroreactivity. Women, current smokers showed significantly less seroreactivity to L antigens as compared with the non-smokers. HIV-positive women showed significantly less (66.7%) antibody response as compared with HIV-negative women (89.3%), with particularly impaired response to L antigens. Women, HIV-positive and current smokers, showed by far the lowest seroprevalence (33.3%) as compared to 75.9% among all other women (OR = 0.158; 95%CI 0.036-0.695, p = 0.014; Fisher's exact test). Importantly, this association did not loose its significance when controlled for confounding from age (continuous variable) in multivariate analysis or using Mantel-Haenszel test for age-groups. CONCLUSION: It is tempting to speculate that HIV-positive current smokers comprise a special high-risk group, with highly impaired immunological response that could prevent eradication of persistent HPV infections and thus contribute to development of CIN3/CC.

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.

BACKGROUND: Human 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. METHODS: The 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. RESULTS: Most 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. CONCLUSION: This 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.

Over-expression of topoisomerase IIalpha is related to the grade of cervical intraepithelial neoplasia (CIN) and high-risk human papillomavirus (HPV), but does not predict prognosis in cervical cancer or HPV clearance after cone treatment.

OBJECTIVE: One of the pathways leading to cervical cancer is a loss of normal cell cycle control. Topoisomerase IIalpha and IIbeta are important nuclear proteins controlling the G2/M checkpoint, and shown to be over-expressed in many human cancers. Their links to oncogenic human papillomavirus (HPV) types and their prognostic value in cervical cancer are practically unexplored. MATERIAL AND METHODS: As part of our HPV-PathogenISS study, a series of 150 squamous cell carcinomas (SCC) and 152 CIN lesions were examined using immunohistochemical (IHC) staining for topoisomerase IIalpha (topo IIalpha), and tested for HPV using PCR with three primer sets (MY09/11, GP5/GP6, SPF). Follow-up data were available from all SCC patients, and 67 CIN lesions had been monitored with serial PCR for HPV clearance/persistence after cone treatment. RESULTS: Topo IIalpha expression increased with increasing grade of CIN (p = 0.0001), with the most dramatic up-regulation upon progression from CIN2 to CIN3 and peaking in SCC (OR 16.23; 95%CI 7.89-33.38). Topo IIalpha up-regulation was also significantly associated with HR-HPV detection in univariate analysis (OR = 3.07; 95%CI 1.70-5.52), but was confounded by the histological grade (Mantel-Haenszel common OR = 1.622; 95%CI 0.782-3.365), and by entering both p16(INK4a) (9) and Survivin (33) in the multivariate regression model. Topo IIalpha did not predict clearance/persistence of HR-HPV after treatment of CIN, and it was not a prognostic factor in cervical cancer in either univariate or multivariate analysis. CONCLUSIONS: Over-expression of topo IIalpha is significantly associated with progression from CIN2 to CIN3, being a late marker of cell proliferation. Its close association with HR-HPV is plausibly explained by the fact that E7 oncoproteins of these HR-HPV (but not LR-HPV) block the normal pRb-mediated inhibition of topo IIalpha by degrading the wild-type Rb.

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.

Recombinant protein-based ELISA and immuno-cytochemical assay for the diagnosis of SARS.

A new Coronavirus (SARS-CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in-house ELISA based on SARS-CoV recombinant antigens was developed. The SARS-CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine-tagged proteins. Six reference sera from SARS patients were used to detect virus-specific IgG in an ELISA using each recombinant protein as coating antigen. High-titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non-SARS, low-tract respiratory infections. Specific hyper-immune sera to SARS-CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS-CoV infected and uninfected cells.