Distribution and Clearance of Adenovirus from the Respiratory Tract

A. Clinical Aspects of Natural Adenoviral Infection in Humans

Adenovirus is an important respiratory pathogen affecting individuals of all ages with an annual incidence of between 5 to 10 million in the United States. Infections can occur sporadically, epidemically and nosocomially but most individuals are infected at a young age; adenovirus accounts for 7 to 10% of all respiratory illnesses in infants and children [8, 9]. Although adenovirus frequently causes a mild, acute upper respiratory illness, e.g., the "common cold," respiratory infections occur as a broad spectrum of distinct clinical syndromes ranging from self-limited acute pharyngitis to fatal pneumonia [10-12], Adenovirus has also been identified an etiological factor of exacerbations in individuals with chronic obstructive lung diseases and infections can be especially problematic in immunocompromised individuals. Examples of the latter include persistent bladder infections in individuals with chemotherapy-induced neutropenia, fatal pneumonia in neonates, and exacerbation of graft rejection and bronchiolitis obliterans in lung transplant recipients [13, 14].

Natural adenovirus infections are typically initiated by deposition of aerosol droplets containing adenovirus on the mucosal surface of the respiratory epithelium [15]. Some virions then diffuse to the cell surface and enter by receptor-mediated endocytosis. Once inside the cell nucleus, wild-type (replication competent) adenovirus DNA overtakes, reprograms, and eventually kills the infected cell ultimately releasing up to 10,000 virions per infected cell. Newly replicated and released virions then infect neighboring cells, repeating the process and thus spreading the infection through the epithelial sheet. The ensuing clinical course is determined by the race between virus replication and spread and the successful mounting of host innate and adaptive immune responses.

B. Distribution of Recombinant, Replication-Deficient Adenoviral Vectors

In considering infection of the respiratory tract by recombinant adenoviruses, it is important to recognize two important and fundamental differences from natural infections by wild-type adenovirus. First, recombinant human adenoviral vectors used to date for in vivo gene transfer have generally been deleted of El region sequences and are thus relatively replication-deficient in human cells (reviewed in [16, 17]). Further, in mice and primates, two species most commonly used in preclinical gene therapy studies, human adenoviral vectors have a species-related host range restriction that prevents viral replication [15]. These restrictions on viral replication, however, do not affect the infection of a given cell by the vector, i.e., virion internalization and transgene expression proceed normally. Second, and very importantly, there is an enormous difference in the size of the infecting inoculum of adenovirus in the two scenarios. A typical natural infection is thought be caused by less than 1000 adenovirus virions. In contrast, human trials have been conducted in which the virus dose administered was up to one billion times higher (~1012 virions/individual).

Studies in rodent models utilizing transtracheal, liquid bolus administration of adenovirus demonstrated that: (1) gene transfer to and expression in airway epithelium was dependent on the dose of virus administered; (2) all cell types could be infected; and (3) gene transfer occurred throughout the bronchial tree but was patchy [18, 19]. Studies in nonhuman primates utilizing bronchoscopic or aerosol delivery also demonstrated gene transfer but confirmed the overall inefficiency of gene transfer [20-23]. Finally, both direct liquid-based or aerosol-based vector administration in humans also demonstrated only low level infection and transduction of the respiratory epithelium. In vitro studies demonstrated, unexpectedly, that while intact airway epithelium was poorly transduced, damaged epithelium, immature epithelial cells, or differentiating airway epithelium were all easily transduced. These findings were reconciled by studies utilizing a bronchial epithelial cell xenograft model which demonstrated that integrin a.v$s, a coreceptor required for adenoviral virion internalization, was not expressed on the apical membrane of mature airway epithelium and was expressed only on the basolateral surface of the cell [24], In the context that physical access to the basolateral membrane of epithelial cells of intact epithelium is restricted by tight junctions which connect adjacent airway epithelial cells just below the apical (luminal) surface [25], this data provided early insight into one mechanism by which airway epithilium present an innate immune barrier to adenovirus infection. The apical membrane surface glycocalyx represents another barrier [26]. Evaluation of various organs of cotton rats or monkeys following intrapulmonary adenovirus vector administration using a sensitive polymerase chain reaction technique demonstrated that vector does not escape from the lung [21, 27]. Recently, studies in rodents have shown that a large portion of adenovirus administered to the respiratory tract is distributed to alveolar macrophages rapidly after pulmonary administration (see below).

C. Kinetics and Mechanisms of Clearance of Adenovirus

The initial report of adenovirus-mediated, in vivo transfer of CFTR to the lungs of cotton rats demonstrated the presence of adenoviral vector DNA in the lung as late as 6 weeks after vector administration [28]. Subsequent studies carried out in various rodent models, nonhuman primates, and humans, however, have demonstrated that most of the adenoviral vector DNA initially administered to the respiratory tract is eliminated from the lung within several weeks in the context of an intact immune system (reviewed in [29]). Data showing that adenovirus-mediated pulmonary transgene expression in athymic mice lasted for more than 3 months implicated the cell-mediated adaptive immunity in pulmonary clearance of adenovirus [30]. This conclusion was supported by the demonstration of prolonged transgene expression in mice depleted of CD4+ cells. The mechanism of this T lymphocyte-mediated clearance was shown to be direct lysis of adenovirus vector-transduced cells by cytotoxic lymphocytes (CTLs) directed at both adenoviral- and transgene-derived proteins. Adaptive immune CTL-mediated clearance is a delayed mechanism because CTL are detected only after several days and are most abundant for 7 to 14 days after infection.

The importance of innate immunity in clearance of adenovirus from the lung was first demonstrated by the finding that ~70% of the adenoviral DNA present immediately after pulmonary administration in mice was eliminated by degradation within 24 h [31] (Fig. 1). Since this "early phase" clearance was well before a significant adaptive immune response could have been mounted, an innate immune mechanism was sought. Similarity in the pattern of clearance in athymic and normal mice demonstrated independence from lymphocyte-based adaptive mechanisms and alveolar macrophages were postulated to be the mechanism of this early clearance. Several findings support this hypothesis. First, in vitro studies demonstrated that infection of human, rat, and murine alveolar macrophages led to loss of approximately two-thirds of the viral DNA within 24 h, whereas similar infection of epithelial cells resulted in no significant loss of viral DNA. Second, pretreatment of the lungs with clodronate-laden liposomes to deplete phagocytic cells significantly impaired the rapid clearance of adenovirus. However, because rapid and significant neutrophil influx occurs during the first 24 h of infection [32], thus overlapping

Acute

0 4 8 12 16 20 24 28 Time after infection (days)

Figure 1 Clearance of viral DNA after adenovirus infection of the respiratory tract. Adenoviral vector DNA is cleared from the lung in a biphasic pattern. Most adenoviral DNA is cleared from the lung very early over the first 24 h after lung infection. The remainder is cleared more slowly over the following several weeks. Clearance during the acute phase is due to internalization and degradation within phagocytes, mostly resident alveolar macrophages (primary clearance). Clearance during the intermediate phase is probably mostly due to recruitment and activation of innate immune NK cells. Clearance during the late phase is principally mediated by the cytotoxic T-cell response. Thus, viral clearance is due to both innate (acute, intermediate) and adaptive (late) immune mechanisms.

the early phase of adenovirus elimination [31], neutrophil-mediated clearance cannot be excluded as an important mediator of viral clearance in these experiments. This concern is supported by the direct demonstration of the uptake of fluorescently labeled adenovirus by neutrophils recruited to the lung using con-focal microscopy (Zsengeller and Trapnell, unpublished observations). Third, infectious, fluorescently labeled adenovirus is rapidly internalized by alveolar macrophages in vivo as early as 1 min following pulmonary administration in mice [33].

The mechanism by which alveolar macrophages internalize adenovirus in vivo is not known but may involve endocytosis and/or phagocytosis and may involve other factors within the local milieu. In order to better understand this mechanism, it is useful to first consider the mechanism of adenovirus infection in epithelial cells which has been well studied (reviewed in [34]). The virion is internalized by receptor-mediated endocytosis and can be summarized as follows: (i) high-affinity binding of the virion to the cell mediated by attachment of the adenovirus fiber knob to its 46-kDa cell surface receptor, CAR [35]; (ii) receptor clustering and rapid virion internalization via a clathrin-coated vesicle mediated by interaction of the adenovirus penton base with integrins av^5 or avP3 [36-40]; (iii) release of clathrin to generate an endocytotic vesicle; (iv) endosome acidification mediated by an endogenous vesicular membrane proton pump [41]; (v) penetration of the endosome membrane (endosome lysis) and release of the virion into the cytoplasm mediated by the TVD motif-containing cytoplasmic tail portion of integrin P,- [42]; (vi) virion translocation to the nuclear membrane mediated by microtubules [39, 43]; (vii) virion hinging to the nuclear pore [44]; (viii) capsid disassembly (continued) at the nuclear pore [34]; and (ix) translocation of viral chromatin into the nucleus through the nuclear pore [44]. Adenovirus uptake by mononuclear phagocytes has been studied to some extent in vitro. In contrast to highly susceptible Coxsackie and adenovirus receptor (CAR)+ epithelial cells, hematopoietic lineage cells including alveolar macrophages, monocytes, and related cell lines do not express CAR and internalize adenovirus about 100 to 1000-fold less well [45-48]. Internalization of adenovirus by these cells in vitro requires cell surface integrin av, similar to CAR+ epithelial cells and upregulation of integrin av|3j and av^3 on human monocytes facilitates infection [45]. Studies using RAW264.7 murine macrophages have shown that the internalization of adenovirus by these cells is temperature-sensitive and calcium-dependent and requires phosphatidylinosi-tol 3-OH kinase [33]. Data from administration of adenovirus to mice in vivo suggest the potential involvement of other factors or an alternative mechanism of uptake. For example, in vivo uptake of adenovirus by alveolar macrophages is reduced in mice deficient in surfactant protein A [49]. Mice deficient in GM-CSF due to targeted gene ablation are unable to clear adenovirus from the lung and alveolar macrophages in these mice are unable to internalize adenovirus efficiently [50] due to a generalized defect in phagocytosis/endocytosis [51]. In contrast, mice deficient in M-CSF (osteopetrotic mice) have no apparent defect in uptake of adenovirus (Zsengeller and Trapnell, unpublished observations). Thus, further studies will be required to determine the mechanism by which alveolar macrophages internalize and degrade adenovirus in vitro and in vivo.

A second important innate immune mechanism of clearance of adenovirus DNA has recently been demonstrated to be the clearance of virus-transduced cells by recruited NK cells [52]. Intravenous administration of adenovirus results in detectable levels of NK cells in infected tissues by 7-10 days and depletion of NK cells prolonged the duration of transgene expression. Interestingly, variation in transgene expression between different strains of mice was associated with significant differences in levels of IL-12 and IFNy production and NK cell activation.

In summary, multiple innate immune barriers block adenovirus infection of the lung and several, redundant innate immune mechanisms of clearance contribute to elimination. Barriers to uptake include production of a mucous layer that traps virions, the ciliary escalator that ejects trapped virions, the epithelial cell glycocalyx that traps virions, and epithelial tight junctions that sequester required adenovirus cell surface receptors away from the luminal surface of the airway epithelial cells. Innate immune mechanisms of clearance include rapid phagocyte-mediated internalization and destruction of the virion (i.e., by primary alveolar macrophages and secondarily recruited neutrophils) and NK cells that destroy adenovirus-infected cells.

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