Host Responses

Another important concept that emerged from in vivo studies in experimental animals was the short duration of transgene expression mediated by Ad vectors. Typically, transgene expression levels peak in 1 to 7 days and decline rapidly to undetectable levels by 2 to 4 weeks (Fig. 5). This is true for most routes of administration with the exception of direct injection into a few immunoprivileged tissues such as brain (66-68). Immediately upon administration, the innate immune system serves to eliminate a large amount of vector. Using viral DNA levels as a means to monitor viral clearance from the liver, approximately 90% of an intravenous bolus is cleared after 24 hr (69). Inhibitors of the reticuloendothelium system reduce this early loss of vector, suggesting that macrophages are responsible for early vector clearance (70).

It is known that infection by wild-type human adenoviruses results in a strong immune response in experimental animals and humans. It was not clear whether replication-deficient

Ad2[Jglu or

Ad2pglu Adößglu

Ad2[Jglu or

Ad2pglu Adößglu

0 1 2 3 7 14151617 21

Figure 5 Quantification of p-glucuronidase expression in the lung over time following repeat administration of the same serotype vector or a vector from an alternate serotype. P-Glucuroni-dase expression in the lung after initial intratracheal administration of Ad2pglu (1011 particles), followed 14 days later by intratracheal administration of either the same vector (Ad2pglu, 109 pfu, o) or a vector of the alternate serotype [Ad5pglu, 109 pfu, • (71)].

0 1 2 3 7 14151617 21

Figure 5 Quantification of p-glucuronidase expression in the lung over time following repeat administration of the same serotype vector or a vector from an alternate serotype. P-Glucuroni-dase expression in the lung after initial intratracheal administration of Ad2pglu (1011 particles), followed 14 days later by intratracheal administration of either the same vector (Ad2pglu, 109 pfu, o) or a vector of the alternate serotype [Ad5pglu, 109 pfu, • (71)].

gene therapy vectors would have the same effect because the net expression of viral genes would be so much lower and the tissues involved would be different from those involved in natural infections. In practice, both cellular and humoral antivector immune responses are observed in rodents after intratracheal (71-73), intravenous (74,75), and intraperitoneal (34) administration. Antibodies against various adenoviral proteins including hexon are induced that can be detected by Western blotting and neutralizing assays. Antivector cytotoxic T lymphocyte (CTL) responses are also observed. The CTL are assumed to eliminate cells infected by the vector in vivo.

When a transgene is used that is foreign to the host, CTL and antibodies are usually, but not always, detected against the expression product of transgene (50,76,77). The basis for this is not fully understood. In classical antigen presentation pathways, a cellular immune response would result from antigens expressed in the antigen-presenting cell and presented in the context of MHC class I. This would imply that Ad infect antigen-presenting cells directly, and there is good evidence both in vivo and in vitro that this occurs (78). But in vivo there is also a strong humoral antitransgene response to many proteins that would not be expected to be taken up by antigen-presenting cells and presented by the class II-depen-dent pathway. It is possible that apoptosis of Ad-infected cells, followed by uptake of apoptotic cells by antigen-presenting cells, is critical (cross-presentation). The strong antitransgene product response has proven useful in using Ad as a carrier for genetic vaccination in situations where both cellular and humoral immunity is desired as described below.

These observations lead to the hypothesis that the immune response is essential for the elimination of adenoviral vectors. Although the initial rapid phase of Ad clearance attributed to the innate immune system was equally rapid in immunodefi-cient mice and immunocompetent mice (79), overall duration of gene expression has been shown to be much longer in immunodeficient mice (79-81). Practically, this suggests ways in which a partial, transient deficiency in the immune system might be exploited to prolong the expression of a therapeutic gene. Conventional immunosuppressants such as corti-costeroids (82) and cyclosporin (83-85) have been used to reduce anti-Ad immune responses and increase the duration of transgene expression in experimental animals. The danger of applying this to human is that opportunistic infections or infection from contaminating wild-type adenovirus may result. But greater persistence of Ad vectors can also be achieved via simultaneous systemic administration of molecules such as antibodies against CD40 ligand (86,87) and CTLA4Ig (88,89), which block interaction between T cells and antigen-presenting cells. These immunomodulators could be used locally, possibly coexpressed on the same adenovirus as the therapeutic gene, to provide more specific immunosuppression.

The humoral and cellular responses also evoke immuno-logical memory, which prevents effective gene expression following subsequent administration of the same vector (Fig. 5). Neutralizing antibodies sequester the readministered vector before it infects cells and cause its immune clearance. Thus, the barriers to readministration of a second vector should be serotype-specific, a concept that has been proven in experimental studies (Fig. 5) (71). Cellular immune system memory can also eliminate any readministered vectors that escape neutralization and infect host cells. The determinants of the cellular immune response are more conserved between serotypes and a second vector of different serotype is eliminated faster from immune animals than from naive animals (Fig. 5).

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