Improved gene delivery to intestinal mucosa by adenoviral vectors bearing subgroup B and d fibers

Abstract

A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses.

FIG. 1.

Interaction of fiber-pseudotyped Ads with the human intestinal epithelial cell line, Caco-2. (A) Binding to undifferentiated Caco-2 cells was measured after a 2-h incubation with 2,500 viral genomes/cell at 4°C and staining of bound viral capsids by flow cytometry. (B) Attachment to a polarized Caco-2 monolayer was performed at 4°C for 2 h with 10⁹ viral genomes per filter and quantified by real-time PCR on viral and cellular DNA. The results are expressed as the ratio of viral and cellular genomes. (C) Transduction of a polarized Caco-2 monolayer was assessed by luminometric measurement of the luciferase transgene activity 2 days after viral infection with 10⁹ viral genomes per filter. The luciferase activity is expressed after normalization for protein concentration for the different fiber-pseudotyped Ads. The figures depict representative results of at least two independent experiments performed in duplicate. Means ± the standard error of the mean (SEM) are represented. Statistically significant differences with Ad5F5 are indicated: ✽, P < 0.05; ✽✽, P < 0.01.

FIG. 2.

Transduction of a polarized murine intestinal epithelium composed of mICcl2 cells by fiber-pseudotyped Ads. Transduction was measured by luminometric measurement of the luciferase transgene activity 2 days after viral infection with 10⁹ viral genomes per filter. Luciferase activity is expressed after normalization for protein concentration for the different fiber-pseudotyped Ads and depicts representative results of at least two independent experiments performed in duplicate. Means ± the SEM are shown. Statistically significant differences with Ad5F5 are indicated: ✽, P < 0.05; ✽✽, P < 0.01.

FIG. 3.

Translocation of viral particles from the apical toward the basolateral pole of the human (A) and murine (B) intestinal epithelium. Medium from the basolateral chamber was sampled for quantification of viral genomes by real-time PCR 2 h and 2 days postinfection with 10⁹ viral genomes per filter. The figures depict representative results of two independent experiments performed in duplicate. Means ± the SEM are shown. Statistically significant differences with Ad5F5 are indicated: ✽, P < 0.05; ✽✽, P < 0.01.

FIG. 4.

Role of sialoglycoconjugates in attachment of fiber-pseudotyped Ads at the surface of a polarized human (A) and murine (B) intestinal epithelium. Binding was quantified by real-time PCR after a 2-h pretreatment of cells with Vibrio cholerae neuraminidase. The results are expressed as the ratio of attachment to treated and untreated cells. The figures depict representative results of at least two independent experiments performed in duplicate. Means ± the SEM are shown. Statistically significant differences between attachment to treated and untreated cells are indicated: ✽, P < 0.05; ✽✽, P < 0.01.

FIG. 5.

Role of CAR (A and B) and CD46 (C and D) in attachment of fiber-pseudotyped Ads at the surface of a polarized human intestinal epithelium. Binding was quantified by real-time PCR either after a 1-h pretreatment of cells with polyclonal anti-CAR (A and B) or anti-CD46 (C) antibodies or 72 h posttransfection with 250 nM CD46-specific siRNAs (D). Transfection efficacy was checked by using fluorescently labeled siRNAs (Block-It Fluorescent Oligo) and specific down regulation of CD46 expression by Western blotting. The results are expressed as the ratio of attachment in the presence of specific (antisera or siRNA) and nonspecific (irrelevant polyclonal antibodies or siRNA) reagents. The figures depict representative results of two independent experiments performed in duplicate. Means ± the SEM are shown. Statistically significant differences between attachment in the presence of specific inhibitory molecules and their respective controls are indicated: ✽, P < 0.05; ✽✽, P < 0.01.

FIG. 6.

Differential expression or localization of CAR (A) and CD46 (B) receptors in Caco-2 and mICcl2 cells. (A) CAR expression was assessed by Western blotting with polyclonal anti-CAR antibodies on lysates of Caco-2 and mICcl2 cells in comparison to lysates of the CAR-negative parental (CHO-pcDNA) and human CAR-positive (CHO-hCAR) CHO cell lines. MW, molecular weight marker. Actin expression levels are also displayed. CAR localization on polarized Caco-2 and mICcl2 cells was observed by confocal microscopy along the xz plane perpendicular to the filter, after staining for CAR (with the fluorescein isothiocyanate and TRITC fluorochromes for Caco-2 and mICcl2 cells, respectively) and the tight junction marker ZO-1 (with the TRITC fluorochrome for Caco-2 cells) (×40 magnification). CAR localization is shown in relation to transduction efficiency for apical and basolateral poles of a CAR-dependent Ad (F40-L) bearing the lacZ gene, as revealed by X-Gal immunohistochemistry and light microscopy (×10 magnification). The proportion of infected cells is indicated in the lower right corner. (B) CD46 expression was assessed by Western blotting with polyclonal anti-CD46 antibodies on lysates of Caco-2 and mICcl2 cells, in comparison to the positive murine testis lysate. (Inset) Reduced exposure of the Caco-2 lane.