No abstract

Human adenoviruses (HAdVs) are being explored as vectors for gene transfer and vaccination. Human adenovirus type 26 (HAdV26), which belongs to the largest subgroup of adenoviruses, species D, has a short fiber and a so-far-unknown natural tropism. Due to its low seroprevalence, HAdV26 has been considered a promising vector for the development of vaccines. Despite the fact that thein vivosafety and immunogenicity of HAdV26 have been extensively studied, the basic biology of the virus with regard to receptor use, cell attachment, internalization, and intracellular trafficking is poorly understood. In this work, we investigated the roles of the coxsackievirus and adenovirus receptor (CAR), CD46, and αv integrins in HAdV26 infection of human epithelial cell lines. By performing different gain- and loss-of-function studies, we found that αvβ3 integrin is required for efficient infection of epithelial cells by HAdV26, while CAR and CD46 did not increase the transduction efficiency of HAdV26. By studying intracellular trafficking of fluorescently labeled HAdV26 in A549 cells and A549-derived cell clones with stably increased expression of αvβ3 integrin, we observed that HAdV26 colocalizes with αvβ3 integrin and that increased αvβ3 integrin enhances internalization of HAdV26. Thus, we conclude that HAdV26 uses αvβ3 integrin as a receptor for infecting epithelial cells. These results give us new insight into the HAdV26 infection pathway and will be helpful in further defining HAdV-based vector manufacturing and vaccination strategies.IMPORTANCEAdenovirus-based vectors are used today for gene transfer and vaccination. HAdV26 has emerged as a promising candidate vector for development of vaccines due to its relatively low seroprevalence and its ability to induce potent immune responses against inserted transgenes. However, data regarding the basic biology of the virus, like receptor usage or intracellular trafficking, are limited. In this work, we found that efficient infection of human epithelial cell lines by HAdV26 requires the expression of the αvβ3 integrin. By studying intracellular trafficking of fluorescently labeled HAdV26 in a cell clone with stably increased expression of αvβ3 integrin, we observed that HAdV26 colocalizes with αvβ3 integrin and confirmed that αvβ3 integrin expression facilitates efficient HAdV26 internalization. These results will allow further improvement of HAdV26-based vectors for gene transfer and vaccination.

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications.

No abstract

Adenoviruses represent exceptional candidates for wide-ranging therapeutic applications, from vectors for gene therapy to oncolytics for cancer treatments. The first ever commercial gene therapy medicine was based on a recombinant adenovirus vector, while most recently, adenoviral vectors have proven critical as vaccine platforms in effectively controlling the global coronavirus pandemic. Here, we discuss factors involved in adenovirus cell binding, entry, and trafficking; how they influence efficiency of adenovirus-based vectors; and how they can be manipulated to enhance efficacy of genetically modified adenoviral variants. We focus particularly on endocytosis and how different adenovirus serotypes employ different endocytic pathways to gain cell entry, and thus, have different intracellular trafficking pathways that subsequently trigger different host antiviral responses. In the context of gene therapy, the final goal of the adenovirus vector is to efficiently deliver therapeutic transgenes into the target cell nucleus, thus allowing its functional expression. Aberrant or inefficient endocytosis can impede this goal, therefore, it should be considered when designing and constructing adenovirus-based vectors.

Three novel tetracationic bis‐triarylboranes with 3,4‐ethylenedioxythiophene (EDOT) linkers, and their neutral precursors, showed significant red‐shifted absorption and emission compared to their thiophene‐containing analogues, with one of the EDOT‐derivatives emitting in the NIR region. Only the EDOT‐linked trixylylborane tetracation was stable in aqueous solution, indicating that direct attachment of a thiophene or even 3‐methylthiophene to the boron atom is insufficient to provide hydrolytic stability in aqueous solution. Further comparative analysis of the EDOT‐linked trixylylborane tetracation and its bis‐thiophene analogue revealed efficient photo‐induced singlet oxygen production, with the consequent biological implications. Thus, both analogues bind strongly to ds‐DNA and BSA, very efficiently enter living human cells, accumulate in several different cytoplasmic organelles with no toxic effect but, under intense visible light irradiation, they exhibit almost instantaneous and very strong cytotoxic effects, presumably attributed to singlet oxygen production. Thus, both compounds are intriguing theranostic agents, whose intracellular and probably intra‐tissue location can be monitored by strong fluorescence, allowing switching on of the strong bioactivity by well‐focused visible light.

No abstract

Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVβ3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVβ3 expressing Cal27-derived cell clone 2B1, αVβ5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6β4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6β4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6β4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVβ3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6β4 not occurred. Taken together, our results identify a key role for α6β4-containing type II hemidesmosomes in regulating anticancer drug sensitivity.