Helper-dependent adenovirus vectors elicit intact innate but attenuated adaptive host immune responses in vivo

Helper-dependent adenovirus (HD-Ad) vectors with all adenoviral genes deleted mediate very long-term expression of therapeutic transgenes in a variety of animal models of disease. These vectors are associated with reduced toxicity and improved safety relative to traditional early region 1 deletion first-generation Ad (FG-Ad) vectors. Many studies have clearly demonstrated that FG-Ad vectors induce innate and adaptive immune responses in vivo; however, a comprehensive analysis of host immune responses to HD-Ad vectors has not yet been performed. In DBA/2 mice, intravenous injection of HD-Ad vectors encoding LacZ (HD-AdLacZ) or a murine secreted alkaline phosphatase (HD-AdSEAP) induced an early expression of inflammatory cytokine and chemokine genes in the liver, including interferon-inducible protein 10, macrophage inflammatory protein 2, and tumor necrosis factor alpha, and were expressed in a pattern similar to that induced by FG-Ad vectors encoding AdSEAP. Like AdSEAP, and consistent with the pattern of cellular gene expression, HD-AdLacZ and HD-AdSEAP induced the recruitment of CD11b-positive leukocytes to the transduced liver within hours of administration. AdSEAP also induced a second phase of liver inflammation, consisting of inflammatory gene expression and CD3-positive lymphocytic infiltrates 7 days posttransduction. In contrast, beyond 24 h no infiltrates or expression of inflammatory genes was detected in the livers of mice receiving HD-AdSEAP. Despite the lack of liver inflammation at 7 days, Ad-specific cytotoxic T lymphocytes could be detected in mice receiving HD-AdSEAP. This lack of liver inflammation was not due to reduced transduction since levels of transgene expression and the amounts of vector DNA in the liver were equivalent in mice receiving HD-AdSEAP and AdSEAP. These results demonstrate that HD-Ad vectors induce intact innate but attenuated adaptive immune responses in vivo. Adenovirus (Ad) vectors are used clinically and experimentally for gene therapy (reviewed by Amalfitano and Parks in reference 1). The majority of gene therapy studies utilize first-generation Ad (FG-Ad) vectors that are rendered replication defective due to the deletion of early region 1 (E1). Although these vectors cannot replicate, FG-Ad vectors induce strong host innate and adaptive immune responses (10). The induction of adaptive immunity by FG-Ad is characterized by the generation of Ad-specific major histocompatibility complex class I-restricted CD8+ cytotoxic T lymphocytes (CTL). In immunocompetent hosts, this response limits the duration of transgene expression and ultimately results in Ad vector clearance within several weeks of administration. The ongoing expression of Ad genes borne by these vectors contributes significantly to the induction of adaptive immunity (31-33). In recent years, a variety of strategies have been developed to minimize or eliminate Ad gene expression in this vector system. Currently, among the most effective vehicles for Ad-mediated gene therapy are helper-dependent Ad (HD-Ad) vectors. HD-Ad vectors lack all viral protein coding sequences and contain only the cis-acting elements required to replicate and package the vector DNA (approximately 500 bp of Ad sequence). Therefore, HD-Ad vectors can accommodate up to 36 kb of foreign DNA. Helper Ads provide the necessary viral genes in trans to package the vector DNA but cannot themselves be packaged due to Cre- or Flp-mediated excision of the helper virus packaging signal (16, 19, 27). In animal models, HD-Ad vectors exhibit stable, long-term transgene expression (8, 23) and are associated with reduced toxicity (13, 14, 29, 35). The development of these agents is thus a major advance in the field of Ad-mediated gene delivery. In addition to their impact on adaptive immunity, FG-Ad vectors also induce innate immune responses (15, 24, 34). Unlike adaptive responses, the induction of innate immunity is triggered by the Ad particle and does not require viral gene expression (11, 24). Within minutes of delivery in vivo, UV-psoralen-inactivated (i.e., transcription-defective) FG-Ad vectors induce the expression of host cytokines and chemokines, similar to transcription-competent FG-Ad vectors (9, 11, 12, 15, 24), resulting in the rapid loss of vector independent of the adaptive immune system (30). Furthermore, transcription-defective FG-Ad vectors can induce Ad-specific CTL, illustrating that adaptive immune mechanisms remain in play despite the apparent lack of viral gene expression (7, 11, 22). Although HD-Ad vectors might be expected to exhibit similar responses, FG-Ad vectors inactivated by UV-psoralen-treatment do not truly mimic HD-Ad vectors, and a detailed analysis of the host immune response to HD-Ad has yet to be performed. We have developed a model of adenoviral gene transfer to study both innate and adaptive immune responses in vivo (11). In this model, intravenous administration of FG-Ad vectors produces a biphasic immune response in the liver of naïve mice. First, FG-Ad vectors trigger rapid (<24-h) induction of inflammatory gene expression within the liver, accompanied by leukocyte recruitment and acute hepatic inflammation. This first phase corresponds to the innate immune response and, in the absence of Ad gene transcription, does not persist beyond 24 h. Following the administration of transcription-competent FG-Ad vectors, a second peak of cytokine and/or chemokine gene expression and inflammation occurs within the liver at 5 to 7 days postinjection. This phase, characterized by predominantly lymphocytic infiltrates and the induction of Ad-specific CTL, represents the adaptive immune response (11). In this study, we employ this model to examine the host immune response to HD-Ad vectors in comparison to the response to FG-Ad vectors. We show that HD-Ad vectors induce innate immune responses similar to those induced by FG-Ad vectors and that they elicit attenuated adaptive immunity in vivo. Our results show that HD-Ad vectors provide an immunological advantage over their FG-Ad counterparts.