Development of a low-seroprevalence, αvß6 integrin-selective virotherapy based on human adenovirus type 10.

Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre-existing immunity will be beneficial for future clinical translation. We generated a low-seroprevalence HAdV-D10 serotype vector incorporating an αvß6 integrin-selective peptide, A20, to target αvß6-positive tumor cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low-affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation factor X, potentially eliminating "off-target" hepatic sequestration in vivo. We engineered an A20 peptide that selectively binds αvß6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvß6+ cancer cell lines demonstrated significantly increased transduction mediated by αvß6-targeted variants compared with controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumors. Replication-competent HAdV-D10.A20 demonstrated αvß6 integrin-selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions, and reduced off-target uptake. Incorporation of an αvß6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.

Efficient Intravenous Tumor Targeting Using the αvß6 Integrin-Selective Precision Virotherapy Ad5NULL-A20.

We previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvß6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvß6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvß6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvß6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvß6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvß6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvß6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes.

Diversity within the adenovirus fiber knob hypervariable loops influences primary receptor interactions.

Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are demonstrating promising efficacy as protective platforms against infectious diseases. Significant clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, and receptor usage are currently lacking. Here, we perform structural and biological analysis of the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our findings provide insight into the cell-virus interactions of HAdV-D26/48, with important implications for the design and engineering of optimised Ad-based therapeutics.

Ad5NULL-A20: A Tropism-Modified, αvß6 Integrin-Selective Oncolytic Adenovirus for Epithelial Ovarian Cancer Therapies.

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvß6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues.Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvß6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed.Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvß3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvß6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non-tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20-treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5.Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvß6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215-24. ©2018 AACR.

Pseudotyped αvß6 integrin-targeted adenovirus vectors for ovarian cancer therapies.

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, αvß6 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 (αvß6high). 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 (αvß6high), 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 αvß6-mediated route was demonstrated, even in the presence of pre-existing anti-Ad5 immunity. Consequently, αvß6-targeted Ad vectors may represent a promising platform for local intraperitoneal treatment of ovarian cancer metastases.

Oncolytic Adenovirus: Strategies and Insights for Vector Design and Immuno-Oncolytic Applications.

Adenoviruses (Ad) are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies.

His-tagged norovirus-like particles: A versatile platform for cellular delivery and surface display.

In addition to vaccines, noninfectious virus-like particles (VLPs) that mimic the viral capsid show an attractive possibility of presenting immunogenic epitopes or targeting molecules on their surface. Here, functionalization of norovirus-derived VLPs by simple non-covalent conjugation of various molecules is shown. By using the affinity between a surface-exposed polyhistidine-tag and multivalent tris-nitrilotriacetic acid (trisNTA), fluorescent dye molecules and streptavidin-biotin conjugated to trisNTA are displayed on the VLPs to demonstrate the use of these VLPs as easily modifiable nanocarriers as well as a versatile vaccine platform. The VLPs are able to enter and deliver surface-displayed fluorescent dye into HEK293T cells via a surface-attached cell internalization peptide (VSV-G). The ease of manufacturing, the robust structure of these VLPs, and the straightforward conjugation provide a technology, which can be adapted to various applications in biomedicine.

Incorporation of Peptides Targeting EGFR and FGFR1 into the Adenoviral Fiber Knob Domain and Their Evaluation as Targeted Cancer Therapies.

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 hCAR(low)/EGFR(high) 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.

Genotype considerations for virus-like particle-based bivalent norovirus vaccine composition.

Norovirus (NoV) genogroup I (GI) and GII are responsible for most human infections with NoV. Because of the high genetic variability of NoV, natural infection does not induce sufficient protective immunity to different genotypes or to variants of the same genotype and there is little or no cross-protection against different genogroups. NoV-derived virus-like particles (VLPs) are promising vaccine candidates that induce high levels of NoV-specific humoral and cellular immune responses. It is believed that a bivalent NoV vaccine consisting of a representative VLP from GI and GII is a minimum requirement for an effective vaccine. Here, we compared the abilities of monovalent immunizations with NoV GI.1-2001, GI.3-2002, GII.4-1999, and GII.4-2010 New Orleans VLPs to induce NoV type-specific and cross-reactive immune responses and protective blocking antibody responses in BALB/c mice. All of the VLPs induced comparable levels of type-specific serum IgG antibodies, as well as blocking antibodies to the VLPs used for immunization. However, the abilities of different VLP genotypes to induce cross-reactive IgG and cross-blocking antibodies varied remarkably. Our results confirm previous findings of a lack of cross-protective immune responses between GI and GII NoVs. These data support the rationale for including NoV GI.3 and GII.4-1999 VLPs in the bivalent vaccine formulation, which could be sufficient to induce protective immune responses across NoV genotypes in the two common genogroups in humans.

High serum levels of norovirus genotype-specific blocking antibodies correlate with protection from infection in children.

BACKGROUND: Norovirus is a common cause of acute gastroenteritis in children. Serum immunoglobulin G (IgG) antibodies have been implicated in protection against norovirus-associated gastroenteritis, but the level, specificity, and functionality necessary for protection remain to be elucidated. METHODS: Norovirus-specific IgG antibodies to genogroup II (GII)-4-2010 New Orleans (NO), GII-4-1999, GII-12-1998, GI-1-2001, and GI-3-2002 virus-like particles (VLPs) were determined by enzyme-linked immunosorbent assay in serum samples collected from children who presented to the hospital because of acute norovirus gastroenteritis in 2009-2011. The blocking activity of the antibodies was tested in a surrogate neutralization assay. RESULTS: Most norovirus infections (62.8%) in the study population were caused by a GII-4 NO variant. Children who acquired GII-4 NO infection had a low preexisting type-specific IgG level and blocking activity of the sera, in contrast to children infected with other GII genotypes. Following GII-4 NO infection, genotype-specific seroconversion and a corresponding increase in blocking antibody potential was observed. Although seroconversion to the heterologous GII-4-1999 variant was observed, there was no corresponding increase in the specific blocking antibody titer. There was no concomitant seroconversion against GI VLPs, indicating a highly genogroup-specific antibody response. CONCLUSIONS: High preexisting norovirus genotype-specific serum IgG titers and blocking activity in children indicate protection from norovirus infection in a strain-specific manner.

Comparison of human saliva and synthetic histo-blood group antigens usage as ligands in norovirus-like particle binding and blocking assays.

Blocking of norovirus-like particle binding to their cellular ligands, histo-blood group antigens with immune sera, is considered a surrogate norovirus neutralization assay. We compared human secretor positive saliva and synthetic biotinylated carbohydrates as a source of histo-blood group antigens in binding and blocking assays. Six norovirus capsid-derived virus-like particles belonging to genogroup I (GI-1-2001 and GI-3-2002) and genogroup II (GII-4-1999, GII-4-2010 New Orleans, GII-4-2012 Sydney and GII-12-1998) noroviruses were produced by a recombinant baculovirus expression system and binding profile to saliva type A, B and O and to synthetic antigens (A trimer, B trimer, H type 1, H type 3, Lewis(a) and Lewis(b)) was identified. Good correlation between virus-like particle binding to saliva type A and synthetic A trimer (r = 0.66, p < 0.05) and saliva type B and synthetic B trimer (r = 0.75, p < 0.05) was observed. Binding of each norovirus virus-like particle to the selected histo-blood group antigens was blocked by convalescent sera from NoV-infected subjects or type-specific mouse antisera. Our results support the use of either saliva or synthetic antigens in blocking assay to measure the ability of norovirus antisera to block virus-like particle binding to the carbohydrate ligands.

Retargeting adenovirus serotype 48 fiber knob domain by peptide incorporation.

Adenovirus type 5 (Ad5) is a commonly used vector for gene therapy, but its efficacy is limited by high seroprevalence and off-target hepatic and splenic sequestration. In order to circumvent these limitations, the use of vectors derived from rare species adenoviruses is appealing. The opportunity to retarget rare species vectors to defined cell types through the incorporation of peptide ligands would be advantageous, particularly in targeting tumors and disseminated metastases. We used predictive structural modeling to assess the CD, DG, HI, and IJ loops of the Ad48 fiber knob and identify optimal incorporation locales for the 20-mer peptide, A20FMDV2 (A20). A20FMDV2 targets ανß6 integrin, which is overexpressed in human carcinomas. Recombinant Ad48 fiber knob proteins Knob48, Knob48-CD-A20, Knob48-DG-A20, Knob48-HI-A20, and Knob48-IJ-A20 were engineered and purified after expression in Escherichia coli. We confirmed that Knob48, Knob48-CD-A20, and Knob48-IJ-A20 formed stable homotrimers. However, Knob48-DG-A20 and Knob-HI-A20 failed to form a trimer. All A20-modified knob proteins blocked the transduction of Ad5-EGFPA20 via ανß6, demonstrating that the inserted A20 peptide was functional. In conclusion, we show that the CD and IJ loops of Ad48 represent suitable sites for targeting peptide incorporation. Interestingly, in vitro gene transfer mediated by the non-factor-X-binding Ad48 vector was not sensitive to immunoglobulins and complement when incubated in the presence of mouse serum, unlike Ad5. These data support the future generation of the corresponding Ad48 viral vectors, Ad48-CD-A20 and Ad48-IJ-A20, which may offer favorable characteristics for targeted delivery in vivo.

Glycosylations in the globular head of the hemagglutinin protein modulate the virulence and antigenic properties of the H1N1 influenza viruses.

With the global spread of the 2009 pandemic H1N1 (pH1N1) influenza virus, there are increasing worries about evolution through antigenic drift. One way previous seasonal H1N1 and H3N2 influenza strains have evolved over time is by acquiring additional glycosylations in the globular head of their hemagglutinin (HA) proteins; these glycosylations have been believed to shield antigenically relevant regions from antibody immune responses. We added additional HA glycosylation sites to influenza A/Netherlands/602/2009 recombinant (rpH1N1) viruses, reflecting their temporal appearance in previous seasonal H1N1 viruses. Additional glycosylations resulted in substantially attenuated infection in mice and ferrets, whereas deleting HA glycosylation sites from a pre-pandemic virus resulted in increased pathogenicity in mice. We then more directly investigated the interactions of HA glycosylations and antibody responses through mutational analysis. We found that the polyclonal antibody response elicited by wild-type rpH1N1 HA was likely directed against an immunodominant region, which could be shielded by glycosylation at position 144. However, rpH1N1 HA glycosylated at position 144 elicited a broader polyclonal response able to cross-neutralize all wild-type and glycosylation mutant pH1N1 viruses. Moreover, mice infected with a recent seasonal virus in which glycosylation sites were removed elicited antibodies that protected against challenge with the antigenically distant pH1N1 virus. Thus, acquisition of glycosylation sites in the HA of H1N1 human influenza viruses affected not only their pathogenicity and ability to escape from polyclonal antibodies elicited by previous influenza virus strains but also their ability to induce cross-reactive antibodies against drifted antigenic variants.