Immune Responses to Reporter Proteins and High Viral Dose Limit Duration of Expression with Adenoviral Vectors: Comparison of E2a Wild Type and E2a Deleted Vectors

Abstract

Experiments designed to evaluate the effect of deletion of E2a on duration of expression using adenoviral vectors led to a series of observations regarding host responses to adenoviral vectors and reporter proteins. In studies using human α₁-antitrypsin (hAAT) as a reporter gene, we found that the duration of expression is very brief for C3H/J and CBA/J mice but is prolonged for C57BL/6J mice, that disappearance of hAAT from the blood is correlated with the appearance of antibodies, and that immunization against hAAT can prevent appearance of the protein in the blood after administration of an adenoviral vector. Deletion of E2a in hAAT vectors did not prolong expession in C3H/J or CBA/J mice and did not shorten duration of expression in C57BL/6J mice. Using similar vectors expressing Escherichia coli β-galactosidase (β-Gal) in immunocompetent mice, short duration of expression with a β-Gal reporter was remarkably different from the long expression with an identical vector expressing hAAT in C57BL/6J. In the case of vectors expressing hAAT, adenoviral sequences persisted in the liver, and inflammatory responses were minimal compared to vectors expressing β-Gal, where adenoviral sequences disappeared from the liver concomitant with a prominent inflammatory response. The duration of expression of β-Gal in hepatocytes was increased in transgenic mice expressing the reporter in keratinocytes, indicating that host immune responses to the reporter can limit duration of expression. Dosage studies indicated that persistence of expression of hAAT can be markedly decreased by administration of high doses of vector in a manner consistent with a nonimmune-mediated toxicity following injection. These experiments indicate that host responses to reporter genes rather than host responses to adenoviral proteins can be the primary determinant of duration of expression under many experimental conditions. Using adenoviral vectors, host responses to reporter genes can limit duration of expression under many experimental conditions. Future studies should be designed to obviate host immune responses to reporter or therapeutic genes to the extent possible and should be interpreted carefully to distinguish host responses to viral genes versus to reporter genes. An unproved experimental paradigm circumventing host responses to reporter genes is needed to evaluate newer generations of adenoviral vectors for effects on duration of expression. High viral dose also can limit duration of expression, suggesting that a high level of gene expression per vector particle may permit lower doses of vector resulting in increased duration of expression as well as decreased toxicity. Considering the extreme variability in host responses depending on experimental conditions and the likelihood of preexisting immunity in humans, there is a particular need for data from primates and from human subjects in a meaningful therapeutic protocol.