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Oncolytic virotherapies based on adenovirus 5 (Ad5) hold promise as adjunctive cancer therapies; however, their efficacy when delivered systemically is hampered by poor target cell specificity and preexisting anti-Ad5 immunity. Ovarian cancer represents a promising target for virotherapy, since the virus can be delivered locally into the peritoneal cavity. Both epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) are overexpressed in the majority of human tumors, including ovarian cancer. To generate adenoviral vectors with improved tumor specificity, we generated a panel of Ad5 vectors with altered tropism for EGFR and FGFR, rather than the natural Ad5 receptor, hCAR. We have included mutations within AB loop of the viral fiber knob (KO1 mutation) to preclude interaction with hCAR, combined with insertions in the HI loop to incorporate peptides that bind either EGFR (peptide YHWYGYTPQNVI, GE11) or FGFR1 (peptides MQLPLAT, M*, and LSPPRYP, LS). Viruses were produced to high titers, and the integrity of the fiber protein was validated by Western blotting. The KO1 mutation efficiently ablated hCAR interactions, and significantly increased transduction was observed in hCARlow/EGFRhigh cell lines using Ad5.GE11, while transduction levels using Ad5.M* or Ad5.LS were not increased. In the presence of physiological concentrations of human blood clotting factor X (hFX), significantly increased levels of transduction via the hFX-mediated pathway were observed in cell lines, but not in primary tumor cells derived from epithelial ovarian cancer (EOC) ascites samples. Ad5-mediated transduction of EOC cells was completely abolished by the presence of 2.5% serum from patients, while, surprisingly, incorporation of the GE11 peptide resulted in significant evasion of neutralization in the same samples. We thus speculate that incorporation of the YHWYGYTPQNVI dodecapeptide within the fiber knob domain may provide a novel means of circumventing preexisting Ad5 immunity that warrants further investigation.

Ovarian cancer accounts for >140 000 deaths globally each year. Typically, disease is asymptomatic until an advanced, incurable stage. Although response to cytotoxic chemotherapy is frequently observed, resistance to conventional platinum-based therapies develop rapidly. Improved treatments are therefore urgently required. Virotherapy offers great potential for ovarian cancer, where the application of local, intraperitoneal delivery circumvents some of the limitations of intravenous strategies. To develop effective, adenovirus (Ad)-based platforms for ovarian cancer, we profiled the fluid and cellular components of patient ascites for factors known to influence adenoviral transduction. Levels of factor X (FX) and neutralizing antibodies (nAbs) in ascitic fluid were quantified and tumor cells were assessed for the expression of coxsackie virus and adenovirus receptor (CAR) and CD46. We show that clinical ascites contains significant levels of FX but consistently high CD46 expression. We therefore evaluated in vitro the relative transduction of epithelial ovarian cancers (EOCs) by Ad5 (via CAR) and Ad5 pseudotyped with the fiber of Ad35 (Ad5T*F35++) via CD46. Ad5T*F35++ achieved significantly increased transduction in comparison to Ad5 (P

Encouraging results from recent clinical trials are revitalizing the field of oncolytic virotherapies. Human adenovirus type 5 (HAdV-C5/Ad5) is a common vector for its ease of manipulation, high production titers and capacity to transduce multiple cell types. However, effective clinical applications are hindered by poor tumor-selectivity and vector neutralization. We generated Ad5/kn48 by pseudotyping Ad5 with the fiber knob domain from the less seroprevalent HAdV-D48 (Ad48). The vector was shown to utilize coxsackie and adenovirus receptor (CAR) but not CD46 for cell entry. A 20-amino acid peptide NAVPNLRGDLQVLAQKVART (A20) was inserted into the Ad5. Luc HI loop (Ad5.HI.A20) and Ad5/kn48 DG loop (Ad5/kn48.DG.A20) to target a prognostic cancer cell marker, αvβ6 integrin. Relative to the Ad5.Luc parent vector, Ad5.HI.A20, Ad5.KO1.HI.A20 (KO1, ablated CAR-binding) and Ad5/kn48.DG.A20 showed ~ 160-, 270- and 180-fold increased transduction in BT-20 breast carcinoma cells (αvβ6high). Primary human epithelial ovarian cancer (EOC) cultures derived from clinical ascites provided a useful ex vivo model for intraperitoneal virotherapy. Ad5.HI.A20, Ad5.KO1.HI.A20 and Ad5/kn48.DG.A20 transduction was ~ 70-, 60- and 16-fold increased relative to Ad5.Luc in EOC cells (αvβ6high), respectively. A20 vectors transduced EOC cells at up to ~ 950-fold higher efficiency in the presence of neutralizing ovarian ascites, as compared to Ad5.Luc. Efficient transduction and enhanced cancer-selectivity via a non-native αvβ6-mediated route was demonstrated, even in the presence of pre-existing anti-Ad5 immunity. Consequently, αvβ6-targeted Ad vectors may represent a promising platform for local intraperitoneal treatment of ovarian cancer metastases

Influenza A is a highly contagious respiratory virus whose surface protein hemagglutinin (HA) constantly obtains mutations at its five antigenic sites. These mutations occasionally lead to acquisition of new N-linked glycosylation sites that may alter the antigenicity and virulence of influenza virus by shielding the major antigenic epitopes. Glycosylation sites 71, 142, 144, 172, 177 and 179 have occurred in the HA globular head of the natural seasonal H1N1 influenza A strains and become fixed over time. The immunomodulatory role of these single and multiple additional N-linked glycosylation sites in the recombinant pandemic A/Netherlands/602/09 H1N1 (rWT) background was assessed. Hemagglutination inhibition (HI) assays were performed with mouse and ferret antisera to evaluate the effect of these additional glycosylations on the breadth of humoral immune response. Poor HI activity was observed with antisera from mice infected with the rWT and the recombinant virus glycosylated at site 144 against 14 natural seasonal H1N1 isolates that occupy variable glycosylations and are antigenically distant from the pandemic strain. It was shown that mouse antisera elicited by viruses containing an additional glycosylation at site 144 or 144-172 were capable of cross-reacting with the rWT and a panel of recombinant mutant viruses with single or multiple additional glycosylations. Conversely, antiserum from mice infected with the rWT had a considerable loss in cross-reactivity against the viruses glycosylated at sites 144 or 144-172. However, antisera from ferrets infected with the 144-glycosylated or 144-172-glycosylated viruses showed narrowed cross-reactivity against a panel of glycosylation mutant viruses and were not capable of neutralising the rWT, as measured by microneutralisation assays. Based on the observations with mouse antisera, most of the polyclonal activity seems to be directed against the antigenic site Sa, efficiently masked by glycosylation at site 144. This alters the antigenicity of the 2009 pandemic H1N1 virus by redirecting the focus of polyclonal response to multiple sites around the site Sa. The broad cross-reactivity was shown to be unique to site 144, since the breadth of polyclonal response elicited by other mutant viruses with single or multiple glycosylation sites was narrowed below homologous titres, having no activity against the viruses containing the site 144. Broadening the humoral immune response by incorporation of additional glycosylation sites provides an approach for universal, broadly reactive influenza vaccines. However, further validation is warranted in both animal models. Keywords: 2009 pandemic H1N1 influenza, antigenicity, antigenic site Sa, broadly reactive influenza vaccine, cross-reactivity, hemagglutinin, humoral immune response, influenza A, neutralising antibodies, N-linked glycosylation

Ovarian cancer is the deadliest gynaecological cancer, with less than half of patients surviving five years from diagnosis. The asymptomatic nature of the early disease commonly results in diagnosis at an advanced stage where peritoneal metastases are prevalent, and the disease rapidly develops resistance to platinum-based agents. Innovative treatments are needed to combat this untreatable disease that has a poor prognosis.\ud Adenoviruses (Ad) are versatile gene therapy vehicles that have been studied for six decades. Their wider clinical use has been limited by poor tumour-specificity, pre-existing immunity, and toxicity-inducing off-target delivery. We have generated an Ad5.3D.A20 vector that is re-targeted to an epithelial cancer-specific marker, αvβ6 integrin, and fully de-targeted from native interactions ‒ αvβ3/5 integrins, coxsackie and adenovirus receptor (CAR), and human coagulation factor 10 (FX). Ad5.3D.A20 selectively transduced αvβ6+ epithelial ovarian cancer (EOC) cells in vitro and clinical ovarian ascites-derived EOC cells ex vivo, including in the presence of neutralising anti-Ad antibodies. In vivo, Ad5.3D.A20 exhibited significantly reduced off-target accumulation and transduction of the liver, spleen and lungs, relative to Ad5. Efficacy studies are underway to investigate its oncolytic potential. Furthermore, we explored the potential use of alternative serotypes from the rare seroprevalence subgroup D. A novel Ad10 vector showed improved resistance from neutralisation and lack of FX binding. Chimaeric Ad5 vectors pseudotyped with Ad10, -15, -24, -29, -48 or -53 fiber had reduced interactions with CAR, but no binding to CD46.\ud Our strategy for translation initially is via intraperitoneal delivery of oncolytic vectors, bypassingmany of the barriers presented by systemic delivery, but enabling transduction of disseminated metastases. These exquisitely tumour-targeted vectors may be further modulated to carry therapeutic moieties to complement their direct cell-killing activity via the stimulation of anti-cancer immunity. The generated tropism-modified vectors have significant therapeutic potential for a wide range of immuno-oncolytic applications.