Canine adenovirus vectors for lung-directed gene transfer: efficacy, immune response, and duration of transgene expression using helper-dependent vectors

Abstract

A major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.

FIG. 1.

Efficacy of CAV-2 vectors in lung tissue. (A) Transduction of human pulmonary cell lines with HAd5 and CAV-2 vectors. The transduction efficiency of CAV-2 and HAd5 vectors was assayed in BEAS2B (epithelial, bronchial) and A549 (epithelial, alveolar) cells. Cells were incubated with CAVDsRed or AdGFP (50 infectious particles/cell) and analyzed by flow cytometry 24 h later. (B) Transduction efficiency of well-differentiated human airway epithelia. Bronchial epithelium was reconstituted in vitro from human primary lung cells after culturing at the medium-airway interface. These cells reconstitute a well-differentiated epithelia, with tight junctions and a basolateral and apical surface. Epithelia were infected with 2.5 × 10³ p.p. of either CAVGFP or AdGFP on the apical surface for 30 or 240 min, and cells were analyzed by FACS 72 h postinfection (n = 8). (C) Polarity of human airway epithelium infection with CAV-2. Epithelia were incubated with 2.5 × 10³ p.p. of CAVGFP on either the apical or basolateral surface for 30 min. Flow cytometry analysis was performed 48 h later (n = 4). **, P < 0.01. (D) Coinstillation in the mouse respiratory tract. CAVDsRed and AdGFP (5 × 10¹⁰ p.p.) were codelivered to 6-week-old C57BL/6 mice by i.n. instillation (n = 4). The lungs were recovered 6 days postinfection. The figure shows an example of a bronchiole in cross-section in which epithelial cells were transduced by CAVDsRed and AdGFP. E, epithelium; L, lumen. Bar, 50 μm.

FIG. 2.

Affect of anti-HAd5 humoral immunity on CAV-2 vector transduction efficiency in vivo. (A) C57BL/6 mice (n = 4/group) were primed i.v. or i.n. with AdGFP (5 × 10¹⁰ p.p. i.n. or 2 × 10¹¹ p.p. i.v.) or PBS on days 0 and 21. Mice were then instilled i.n. with either Adβgal or CAVβgal (5 × 10¹⁰ p.p.) on day 30. The mice were sacrificed 6 days later. Lungs were recovered after intracardiac perfusion and bronchoalveolar lavage with PBS. Blood was also collected at days −1, 21, 30, and 36. On day 6, an i.n. primed mouse was sacrificed to confirm the efficacy of AdGFP transduction in the lung. We found EGFP-positive cells throughout the respiratory airways (not shown). Mock priming and i.n. instillations were done with an equivalent volume of 10% glycerol-PBS. (B) Neutralization assays. The level of HAd5-specific or cross-reacting NAb was assayed in the sera obtained from mice immunized with 1 μl i.v. or 2 μl i.n. at day 30. For HAd5-neutralization assay, 911 cells were infected with AdGFP previously incubated with a fixed volume of serum and analyzed by flow cytometry 24 h postinfection. Cells were infected with 10 p.p. of AdGFP/cell; a multiplicity of infection that results in approximately 30% of EGFP-positive cells 24 h postinfection. For cross-neutralization assays, DKCre cells were infected with CAVGFP previously incubated with sera. Human and dog sera containing NAb against HAd5 or CAV-2 were used as controls in each case. The results are shown as the percentages of transduction inhibition of each vector. Each bar represents the mean from the cohort (n = 8). (C) NAb titers from i.n. and i.v. primed mice. 911 cells were infected with AdGFP, incubated with serial dilutions of sera, and analyzed by flow cytometry 24 h postinfection. Relative NAb titers are expressed as the highest reciprocal serum dilution resulting in reduction of transduction by at least 50%. Titers are the mean values of the results from three independent experiments. The bar is the mean titer value for each cohort (n = 8). (D and E) Effect of anti-HAd5 humoral immunity against on CAV-2 transduction efficiency in mouse lung. The level of β-Gal activity in the lung of mice primed i.v (D) or i.n. (E) with AdGFP or PBS and i.n. instilled with Adβgal or CAVβgal. Each bar represents the mean value of the results for four animals ± standard error of the mean. RLU, relative light units; +, present; −, absent; ***, P < 0.001.

FIG. 3.

Effect of NAb on transduction of human airway epithelia with HAd5 and CAV-2. CAVGFP or AdGFP (2.5 × 10³ p.p.) was added to the apical surface of well-differentiated human airway epithelia in normal medium or to epithelia in medium preincubated for 24 h with a 1:100 dilution of human serum. Flow cytometry analysis was performed 72 h postinfection. +, present; −, absent.

FIG. 4.

Helper-dependent CAV-2 vector allows long-term expression of the transgene in the mouse respiratory tract. C57BL/6 mice (n = 3/group) received 5 × 10¹⁰ p.p. of Spike by i.n. instillation. Mice were sacrificed 6 days (A), 3 weeks (B), or 3 months (C) postinfection, and the lungs were recovered after fixation by intracardiac perfusion using 4% paraformaldehyde-PBS. Nuclei are in blue. Bar, 20 μm.

FIG. 5.

CAV-2 vectors are less inflammatory than HAd5 in the mouse respiratory tract. C57BL/6 mice (n = 4/group) received 10¹¹ p.p. of either Adβgal or CAVβgal by i.n. instillation. Mice were sacrificed 3 h, 24 h, or 6 days postinfection, and the BALF were recovered. The inflammation induced by i.n. inoculation was monitored by measuring the TNF-α levels (A) or counting the total cells (B) in mouse BALF. Data are expressed as means ± standard errors of the means. **, P < 0.01; ***, P < 0.001.

FIG. 6.

Effect of FK228 pretreatment on CAV-2 vector transduction efficiency in lungs. (A) BEAS2B and A549 human pulmonary cells were treated with FK228 (0 to 5 ng/ml) for 24 h before infection with CAVGFP (50 or 100 infectious particles/cell, respectively). Cells were analyzed by flow cytometry 24 h postinfection. (B) C57BL/6 mice were treated with FK228 (0 to 50 μg/kg) by i.n. instillation 24 h before i.n. inoculation with CAVβgal (5 × 10¹⁰ p.p.). Lungs were then recovered after intracardiac perfusion with PBS, and β-Gal activity was measured using a luminescent assay and normalized with the concentration of proteins in each homogenate. β-Gal activity in lungs from PBS- and FK228-treated mice was not significantly different (not shown). Each bar represents the mean ± standard error of the mean (n = 4/group). RLU, relative light units; *, P < 0.05.