Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia.
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Recent identification of two receptors for the adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I alpha-2 domain allows the molecular basis of adenoviral infection to be investigated. Earlier work has shown that human airway epithelia are resistant to infection by adenovirus. Therefore, we examined the expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain the relative resistance to adenovirus infection from the apical surface. In contrast, when the virus was applied to the basolateral surface, gene transfer was much more efficient because of an interaction of adenovirus fiber with its receptors. In addition, when the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space where fiber receptors are located increase gene transfer efficiency. (+info)
The human HLA-A*0201 allele, expressed in hamster cells, is not a high-affinity receptor for adenovirus type 5 fiber.
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The coxsackie B virus and adenovirus receptor (CAR) and the major histocompatibility complex (MHC) class I alpha2 domain have been identified as high-affinity cell receptors for adenovirus type 5 (Ad5) fiber. In this study we show that CAR but not MHC class I allele HLA-A*0201 binds to Ad5 with high affinity when expressed on hamster cells. When both receptors are coexpressed on the cell surface of hamster cells, Ad5 fiber bind to a single high-affinity receptor, which is CAR. (+info)
Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector.
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Important therapeutic applications of genetically modified dendritic cells (DC) have been proposed; however, current vector systems have demonstrated only limited gene delivery efficacy to this cell type. By means of bispecific Abs, we have dramatically enhanced gene transfer to monocyte derived DC (MDDC) by retargeting adenoviral (Ad) vectors to a marker expressed on DC, CD40. Adenovirus targeted to CD40 demonstrated dramatic improvements in gene transfer relative to untargeted Ad vectors. Fundamental to the novelty of this system is the capacity of the vector itself to modulate the immunological status of the MDDC. This vector induces DC maturation as demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a MLR. In comparing this vector to other Ad-based gene transfer systems, we have illustrated that the features of DC maturation are not a function of the Ad particle, but rather a consequence of targeting to the CD40 marker. This vector approach may thus mediate not only high-efficiency gene delivery but also serve a proactive role in DC activation that could ultimately strengthen the utility of this vector for immunotherapy strategies. (+info)
Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.
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Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages. (+info)
Molecular determinants of adenovirus serotype 5 fibre binding to its cellular receptor CAR.
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Adenovirus (Ad) tropism is mediated in part through the fibre protein. The common coxsackie B virus and Ad receptor (CAR) was recently identified as the major receptor for subgroup C Ad serotype 5 (Ad5) and serotype 2 (Ad2) fibres. Effects of mutations in the Ad5 fibre gene were studied to assess domains of the fibre capsomer that could alter virus tropism without altering virus assembly and replication. All mutants that accumulated as fibre monomers failed to assemble with a penton base and proved lethal for Ad5 which suggests that the absence of infectious virions resulted in part from a defect in fibre penton base assembly. Cell binding capacity of all fibre mutants was investigated in cell binding competition experiments with adenovirions using CHO-CAR cells (CHO cells that have been transfected with CAR cDNA and express functional CAR). The results suggest that the R-sheet of the Ad5 fibre knob monomer contains binding motifs for CAR and that beta-strands E and F, or a region close to them, may also be involved in receptor recognition. (+info)
Expression of the coxsackievirus and adenovirus receptor in cultured human umbilical vein endothelial cells: regulation in response to cell density.
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Primary cultures of human umbilical vein endothelial cells (HUVEC) express the human coxsackievirus and adenovirus receptor (HCAR). Whereas HCAR expression in HeLa cells was constant with respect to cell density, HCAR expression in HUVEC increased with culture confluence. HCAR expression in HUVEC was not quantitatively altered by infection with coxsackievirus B. (+info)
Picornavirus receptor down-regulation by plasminogen activator inhibitor type 2.
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Therapeutic interference with virus-cell surface receptor interactions represents a viable antiviral strategy. Here we demonstrate that cytoplasmic expression of the serine protease inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), affords a high level of protection from lytic infection by multiple human picornaviruses. The antiviral action of PAI-2 was mediated primarily through transcriptional down-regulation of the following virus receptors: intercellular adhesion molecule 1 (ICAM-1, a cellular receptor for the major group of rhinoviruses), decay-accelerating factor (a cellular receptor for echoviruses and coxsackieviruses), and to a lesser extent the coxsackie-adenovirus receptor protein (a cellular receptor for group B coxsackieviruses and group C adenoviruses). Expression of related cell surface receptors, including membrane cofactor protein and the poliovirus receptor, remained unaffected. These findings suggest that PAI-2 and/or related serpins may form the basis of novel antiviral strategies against picornavirus infections and also therapeutic interventions against ICAM-1-mediated respiratory inflammation. (+info)
Coxsackie and adenovirus receptor (CAR)-dependent and major histocompatibility complex (MHC) class I-independent uptake of recombinant adenoviruses into human tumour cells.
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The role of two receptors, previously proposed to mediate the entry of adenoviruses into human cells, the coxsackie and adenovirus receptor (CAR) and the major histocompatibility complex (MHC) class I heavy chain has been investigated. The expression of MHC class I in many tumours is reduced or absent, therefore if this were a means by which adenoviruses gained entry into cells, it would have important implications for their application in cancer treatment. In order to determine if MHC class I heavy chain is involved in adenovirus type 5 (Ad5) uptake, the binding of recombinant Ad5 fibre knob domain (which mediates viral attachment) to human cell lines that had greatly different levels of surface MHC class I was studied. We also created derivatives of a non-permissive Chinese hamster ovary (CHO) cell line that expressed human class I (HLA-A2) and found that these cells did not bind fibre or take up virus. In addition, the extracellular domain of CAR was expressed in E. coli and used to generate a polyclonal anti-CAR antibody. This antibody blocked both 125I labelled fibre knob binding and virus uptake. Thus CAR, and not MHC class I, is a receptor for human adenoviruses in cultured tumour cells. Tissue CAR levels may therefore be an important factor in the efficiency of adenovirus-mediated gene therapy. (+info)