Canine adenovirus vectors: an alternative for adenovirus-mediated gene transfer

Abstract

Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.

FIG. 1

Generation of CAVGFP. ptGFP was generated by homologous recombination in E. coli BJ5183 by using SwaI-linearized pTG5412 and a 4.7-kb BglII/FspI fragment from pCAVGFP (nucleotide positions are from CAV-2; diagram not drawn to scale). NotI-digested ptGFP was transfected into DK/E1-1 cells and amplified five to six times on E1-transcomplementing cells, and CAVGFP was purified as described in Materials and Methods.

FIG. 2

Digestion and Southern blot analysis of CAVGFP and CAVGFPΔE1A DNAs. (a) Vector or plasmid DNA (500 ng) was digested with EcoRI and NotI (in order to remove the 2-kb pPolyII backbone from the terminal fragments seen in lanes 1, 2, and 4), electrophoresed through a 0.7% agarose gel, and stained with ethidium bromide. Lanes: 1, pTG5412; 2, ptGFPΔE1A; 3, CAVGFPΔE1A; 4, ptGFP; 5, CAVGFP; M, 1-kb DNA ladder (GIBCO). Southern blot analysis was performed by using a fragment of the E1A region (b) or GFP cDNA (c) as the radiolabelled probe. (d) Locations of the EcoRI sites and the fragment sizes in the vectors.

FIG. 3

Quantitative analysis of transduction efficiencies of CAVGFP and AdGFP in human cells. (a) A172, HeLa, and HT 1080 cells were infected with each vector and assayed for GFP expression 48 h posttransduction. Data represent the amount of vector required to generate 10% GFP-positive cells/well, expressed as the number of input particles/well. Data are means from five experiments ± standard deviations. (b) HeLa cells incubated with an increasing number of particles of CAVGFP and AdGFP and analyzed by flow cytometry 24 h posttransduction. Data are the means ± standard deviations from triplicate samples.

FIG. 4

In vivo transduction of the airway epithelia in mice by using CAV vectors. A total of 10¹¹ particles of CAVβgal, CAVGFP, or AdGFP was delivered intranasally in BALB/c mice, and lung sections were assayed 3 or 4 days later. (a through c) Nuclear-localized β-galactosidase activity from CAVβgal, as demonstrated by the blue precipitate, in the proximal (a) and distal (b) airways and in the alveoli (c). (d through g) GFP expression in distal airways from CAVGFP (d and e) and AdGFP (f and g) as shown by phase contrast (d and f) and fluorescence (e and g).

FIG. 4

In vivo transduction of the airway epithelia in mice by using CAV vectors. A total of 10¹¹ particles of CAVβgal, CAVGFP, or AdGFP was delivered intranasally in BALB/c mice, and lung sections were assayed 3 or 4 days later. (a through c) Nuclear-localized β-galactosidase activity from CAVβgal, as demonstrated by the blue precipitate, in the proximal (a) and distal (b) airways and in the alveoli (c). (d through g) GFP expression in distal airways from CAVGFP (d and e) and AdGFP (f and g) as shown by phase contrast (d and f) and fluorescence (e and g).

FIG. 5

Preexisting humoral immunity. Sera from healthy blood bank donors (n = 50) were assayed for the presence of neutralizing CAV-2 antibodies. In this assay only one sample was partially able (∼24%) to inhibit CAVGFP transduction, while 26 of 50 samples completely inactivated AdGFP transduction.