Search

Background The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells. Methods Envisioning clinical application of the best characterized anti-HPV16 E6 and –HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed. Results We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway. Conclusion Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

Human Papillomavirus 16-associated cancer, affecting primarily the uterine cervix but, increasingly, other body districts, including the head&ndash, neck area, will long be a public health problem, despite there being a vaccine. Since the virus oncogenic activity is fully ascribed to the viral E6 and E7 oncoproteins, one of the therapeutic approaches for HPV16 cancer is based on specific antibodies in single-chain format targeting the E6/E7 activity. We analyzed the Complementarity Determining Regions, repositories of antigen-binding activity, of four anti-HPV16 E6 and -HPV16 E7 scFvs, to highlight possible conformity to biophysical properties, recognized to be advantageous for therapeutic use. By epitope mapping, using E7 mutants with amino acid deletions or variations, we investigated differences among the anti-16E7 scFvs in terms of antigen-binding capacity. We also performed computational analyses to determine whether length, total net charge, surface hydrophobicity, polarity and charge distribution conformed well to those of the antibodies that had already reached clinical use, through the application of developability guidelines derived from recent literature on clinical-stage antibodies, and the Therapeutic Antibodies Profiler software. Overall, our findings show that the scFvs investigated may represent valid candidates to be developed as therapeutic molecules for clinical use, and highlight characteristics that could be improved by molecular engineering.

Background Ebola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide. The EBOV pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. The multifunctional viral VP35 protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (IFN-α/β) response, and represents a suitable target for the development of strategies to control EBOV infection. Phage display technology permits to select antibodies as single chain Fragment variable (scFv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. ScFv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scFv is expressed as intracellular antibody (intrabody) or delivered into the cells. Results Monoclonal antibodies (mAb) in scFv format specific for the EBOV VP35 were isolated from the ETH-2 library of human recombinant antibodies by phage display technology. Five different clones were identified by sequencing, produced in E.coli and expressed in CHO mammalian cells to be characterized in vitro. All the selected scFvs were able to react with recombinant VP35 protein in ELISA, one of the scFvs being also able to react in Western Blot assay (WB). In addition, all scFvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in A549 cells showed that two of the scFvs can significantly hamper the inhibition of the IFN-β-induced RIG-I signaling cascade mediated by EBOV VP35. Conclusion Five antibodies in scFv format recognize an active form of EBOV VP35 in ELISA, while one antibody also recognizes VP35 in WB. Two of these scFvs were also able to interfere with the intracellular activity of VP35 in a cell system in vitro. These findings suggest that such antibodies in scFv format might be employed to develop therapeutic molecules able to hamper EBOV infections.

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.

// Carla Amici 1 , Michela Visintin 2 , Francesca Verachi 1,3 , Francesca Paolini 4 , Zulema Percario 5 , Paola Di Bonito 3 , Angela Mandarino 3 , Elisabetta Affabris 5 , Aldo Venuti 4 and Luisa Accardi 3 1 University of Rome Tor Vergata, Department of Biology, Via della Ricerca Scientifica, Rome, Italy 2 Rottapharm Biotech Srl, Biotherapeutics Discovery, Area Science Park – Basovizza, Trieste, Italy 3 Istituto Superiore di Sanita, Department of Infectious, Parasitic and Immunomediated Diseases, Viale Regina Elena, Rome, Italy 4 Regina Elena National Cancer Institute, Laboratory of Virology and HPV-Unit, via delle Messi d’Oro, Rome, Italy 5 University Roma Tre, Department of Science, Viale G. Marconi, Rome, Italy Correspondence to: Luisa Accardi, email: // Keywords : E6 oncoprotein, human papillomaviruses, cancer therapy, scFv, intracellular antibodies Received : September 03, 2015 Accepted : January 04, 2016 Published : January 15, 2016 Abstract Single-chain variable fragments (scFvs) expressed as “intracellular antibodies” (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. Here we use an intrabody targeting the E6 oncoprotein of Human papillomavirus 16 (HPV16) to address the issue of a non-invasive therapy for HPV cancer patients. A scFv against the HPV16 E6 was selected by Intracellular Antibody Capture Technology and expressed as I7nuc in the nucleus of HPV16-positive SiHa, HPV-negative C33A and 293T cells. Colocalization of I7nuc and recombinant E6 was observed in different cell compartments, obtaining evidence of E6 delocalization ascribable to I7nuc. In SiHa cells, I7nuc expressed by pLNCX retroviral vector was able to partially inhibit degradation of the main E6 target p53, and induced p53 accumulation in nucleus. When analyzing in vitro activity on cell proliferation and survival, I7nuc was able to decrease growth inducing late apoptosis and necrosis of SiHa cells. Finally, I7nuc antitumor activity was demonstrated in two pre-clinical models of HPV tumors. C57BL/6 mice were injected subcutaneously with HPV16-positive TC-1 or C3 tumor cells, infected with pLNCX retroviral vector expressing or non-expressing I7nuc. All the mice injected with I7nuc-expressing cells showed a clear delay in tumor onset; 60% and 40% of mice receiving TC-1 and C3 cells, respectively, remained tumor-free for 17 weeks of follow-up, whereas 100% of the controls were tumor-bearing 20 days post-inoculum. Our data support the therapeutic potential of E6-targeted I7nuc against HPV tumors.

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-PathogenISS 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). Follow-up data were available for all SCC patients, and 67 GIN 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 HRHPV 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.

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.

The complete nucleotide sequence of Toscana (TOS) virus (Bunyaviridae, Phlebovirus) L segment was determined. The L segment is 6404 nucleotides long, containing a single open reading frame (ORF) in the viral complementary sense coding for a protein of 2095 amino acids that, as in the case of negative strand RNA viruses, could be part of the RNA polymerase of TOS virus. This ORF is expressed by a messenger RNA (mRNA) as long as the genomic segment. Like the mRNAs expressed by the genomic segments of the other Bunyaviruses, the L mRNA has non-templated sequences at the 5' end. The comparison of TOS L protein sequence with the corresponding sequences of other negative strand RNA viruses showed a very high homology only with the Rift Valley Fever (RVF) virus. The residues conserved between the two proteins are mainly concentrated in the central region and contain three DD motifs proposed by Argos (1988) to be functional domains of DNA and RNA polymerases. The complete sequence of the Toscana virus L genomic segment has been deposited in the EMBL library with the accession number X68414.

Objective Proliferating cell nuclear antigen (PCNA) is essential for DNA replication of mammalian cells and their small DNA tumour viruses. The E7 oncoprotein of high-risk human papillomavirus (HPV) is known to activate PCNA, shown to be up-regulated in CIN and cervical cancer (CC), but still incompletely studied as an intermediate endpoint marker in this disease. Material and methods As part of our HPV-PathogenISS study, a series of 150 CCs and 152 CIN lesions were examined using immunohistochemical (IHC) staining for PCNA, 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 of the CIN lesions had been monitored with serial PCR for HPV after cone treatment. Results Expression of PCNA increased in parallel with the grade of CIN, with major up-regulation upon transition to CIN3 (OR 21.77; 95%CI 6.59–71.94) ( p = 0.0001). Intense PCNA expression was 100% specific indicator of CIN, with 100% PPV, but suffers from low sensitivity (34.8%) and NPV (10.8%). PCNA expression was also significantly associated to HR-HPV with OR 3.02 (95%CI 1.71–5.34) ( p = 0.0001), and this association was not confounded by the histological grade (Mantel–Haenszel common OR = 2.03; 95%CI 1.06–3.89) ( p = 0.033). Expression of PCNA did not predict clearance/persistence of HR-HPV after treatment of CIN, and it was not a prognostic predictor in CC in univariate or in multivariate analysis. Conclusions Up-regulation of PCNA was closely associated with HR-HPV and progressive CIN, most feasibly explained by the abrogation of normal cell cycle control by the E7 ongogene, reverting the p21 Cip1 -mediated inhibition of PCNA. However, the fact that PCNA is also expressed in normal squamous epithelium precludes the use of this marker as a potential screening tool for CC.

The arthropod-borne Toscana virus has been associated with acute neurological disease in humans. In this study, the viral envelope glycoproteins were expressed in soluble form in a baculovirus system. The recombinant sGN and sGC proteins were used as viral antigens in a Western blot assay to analyze the specific immune response in sera from patients with recognized virus-associated aseptic meningitis. The anti-glycoprotein and the anti-nucleoprotein N IgG responses were compared by an immunoassay based on the recombinant proteins. In this system, all the sera showed a high reactivity to the N protein, but they differed in the response to the glycoproteins.

BackgroundHuman 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.MethodsThe 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.ResultsScFv51 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.ConclusionThe characterization of 5 specific anti-HPV16 E7 scFvs shows features important for their activityin 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 activityin vivo.

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.

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.