expression are equivalent to those of AvlCFl infected Hela cells. Importantly, however, it is 
unclear whether this will occur in humans in vivo within the respiratory epithelium because 
infection of freshly isolated explants of human bronchial epithelial cells failed to demonstrate 
immuno-precipitable hexon protein (the most abundantly expressed adenoviral protein) (Crystal 
et.al., RAC proposal, 1992). 
In regard to the potential development of toxicity or of a sublethal host immune response, 
the cotton rat provides a useful model for human respiratory epithelium due to the close 
similarity of the molecular events (with regards to adenovirus) and the respiratory pathology in 
lung tissues of the cotton rats and humans after infection by wild-type human adenovirus. 
Studies in cotton rats have addressed the question of toxicity following administration of 
AE1, AE3 adenovirus to the respiratory epithelium in vivo. Dose response studies show that the 
LD50 for Ad5 (wild-type adenovirus) was in the range of 5 x 10? pfu per animal. In contrast, 
respiratory administration of AvlCFl of up to 5 x 10 10 pfu after in cotton rats resulted in no 
detectable toxicity after more than 50 days (Crystal et.al., RAC proposal, 1992). In another 
animal model, administration of AvlCFl (2 x 10 10 pfu/kg) to a single lung of Rhesus monkeys 
(n=4) resulted in no deaths during the three month study period (Crystal et.al., RAC proposal, 
1992). Similarly, administration of 3 x 10 n pfu of Ad.CB-CFTR was well tolerated in monkeys 
(Wilson et.al., RAC proposal, 1992). 
The morphological assessment of the respiratory epithelium following pulmonary 
administration of AvlCFl to the cotton rat lung (lOMO 10 pfu/kg) demonstrated a patchy 
mononuclear infiltrate in the bronchoalveolar distribution focused in the perivascular space 
(Crystal et.al., RAC proposal, 1992). We have also observed similar pulmonary mononuclear 
infiltrates in preliminary experiments in cotton rat exposed intranasally to 6 x 10 8 pfu of 
AvlLacZ4 or 2 x 10 8 pfu of AvlCF2 (approximately 4 x 10 9 per kg). In the monkey model, 
administration of one of several AE1,AE3 vectors (Ad-CMV-lacZ, Ad.CB-CFTR) the single 
lobe of a Rhesus monkey via bronchoscopy demonstrated no pathology at doses of 3 x 10 u 
pfu/animal (Wilson et.al., RAC proposal, 1992). 
In the context of what may be mild local cellular immune response to respiratory 
administration of AE1 adenoviral vectors, a humoral response also may be elicited. For example 
administration of AvlCFl to cotton rats via the respiratory route resulted in significant anti- 
adenoviral antibody titers as early as one week following exposure (Crystal et.al., RAC 
proposal, 1992) which persisted for up to four months. Studies are still ongoing to determine 
whether these antibodies were neutralizing to adenovirus-CFTR. In contrast, administration of 
AvlCFl to Rhesus monkeys gave a less consistent humoral response (Crystal et.al., RAC 
proposal, 1992). In other experiments, administration of an adenovirus vector to rhesus 
monkeys showed an increase in serum neutralization antibodies to adenovirus after 10 days with 
a fall after 50 days. The titer increased again after repeated administration of the virus. While 
systemic humoral immunity is clearly elicited by respiratory administration of AE1 recombinant 
adenovirus, it is unclear as to the presence and extent of local humoral immunity or local 
cellular immunity. There are no data on whether or not these antibodies may cause any 
hypersensitivity response. Such issues will need to be further evaluated in this protocol and in 
future protocols. 
In summary, in animal models there is evidence of mild inflammation following administra- 
tion of the vector to the lungs of cotton rats, but not in rhesus monkeys. Importantly, rhesus 
monkeys given a single dose of an adenovirus-lacZ construct showed no observable changes in 
behavior, examination, vital signs, CBCs, serum chemistries, arterial blood gases, chest x-rays, 
and lung compliance (Crystal et.al., RAC Proposal, 1992). 
Recombinant DNA Research, Volume 17 
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