Host Response to Gene Therapy Vectors

Three phases have been described in the elimination of adenovirus after intravenous delivery [57]. Phase one involves innate immune mechanisms, which occurs within 24 h postinfection and accounts for the elimination of 90% of the adenoviral DNA. Phase two is mediated by the adaptive immune system and consists of a cytotoxic T lymphocyte (CTL) and/or antibody response to the transgene and/or to the viral proteins. Phase three is characterized by a slow and constant decrease of the transgene expression even in the absence of an immune response to the transgene and is thought to be the result of leaky expression of viral gene products which leads eventually to clearance of the transduced cells.

A. Innate Immune Response

Although immune-related mechanisms play a significant role in eliminating the recombinant Ad genome following in vivo administration of Ad vectors, it is not clear how the immune response to Ad vectors is initiated, i.e., for the immune system to be sensitized to Ad antigens. Studies by Worgall et al. [58] suggest that there is some initial destruction of the Ad vectors by innate immune mechanisms. In contrast to the antigen-specific, adaptive immune response, innate immune mechanisms comprise the immediate, nonantigen-specific events which include tissue macrophages, which act as scavengers to clear incoming pathogens. In addition tissue macrophages influence the initiation of the adaptive immune response. Innate pathways of virus clearance are also mediated by Kupffer cells within the liver [58]. Following the innate immune response a second phase of the immune response occurs, the adaptive immune response. This results in the generation of antibodies against viral capsid proteins and transgene, i.e., the humoral immune response, in addition CTLs against viral proteins and transgene products ensue to generate the cellular immune response.

B. Adaptive Immune Response: B-T Cell Interactions

Activated T cells play a critical role in the generation of humoral and cellular immune responses. In general there are two major classes of T cells, a

Activate Bi ells Activate CTL's

Figure 2 (a) Antigen-presenting cells (APCs) such as dendritic cells and macrophages ingest foreign proteins (viral particles) nonspecifically. Ingested antigens are processed intracellular!/ and presented as peptide fragments in the context of the MHC class II complex. Activation of the helper T cell occurs following recognition of the foreign peptide/MHC class II molecule by the T-celi receptor, TCR. In addition a second signal is required for helper T-cell activation which occurs when the plasma membrane bound signaling molecules B7-1/B7-2 are recognized by a coreceptor protein CD28/CTLA4-lg which is present on the surface of the helper T cell. Cellular immune response: At the same time viral vectors can infect (APCs) and deposit their genomes into the nucleus. The genome encodes viral and transgene proteins that are expressed and presented by MHC class 1 molecules to CD8+ cells or cytotoxic T lymphocytes (CTLs). (b) Foreign antigens are taken up from the extracellular fluid by receptor mediated endocytosis following binding to the B cell receptor. They are then degraded and recycled to the cell surface in the form of peptides bound to MHC class II molecules. Thus the helper T cell activates the B cell that displays the same antigen in the context of the MHC class II molecule as originally activated it. In addition, interaction of CD40 ligand with CD40 activates B cells to proliferate and mature into memory and antibody-secreting cells. In secondary antibody responses, memory B cells themselves may act as APCs and activate helper T cells as well as being the subsequent targets of the helper T cells.

1L-4 IL-5 Interleukins

Figure 2 (continuedj

1L-4 IL-5 Interleukins

Activated Helper T Cell

Figure 2 (continuedj cytotoxic CD8+ T cells (CTLs) and helper T cells (CD4+ T cells). In the context of a viral infection, CTLs eliminate virally infected cells expressing neoantigens such as viral proteins and the transgene product. In contrast, CD4+ T cells help activate the responses to extracellular antigens by stimulating B cells to proliferate and secrete antibodies. Activation of antigen-presenting cells (APCs) and B cells by input viral capsid proteins underlies the mechanism responsible for the production of the humoral immune response to Ad vectors. Administration of UV-inactivated virus leads to a full humoral response without any CTL involvement, which is consistent with the role of exogenous viral capsid proteins in the activation of B cells and of endogenously produced antigens in the activation of primary CD8+ cells [59, 59a]. However, activation of CD4+ T cells by viral capsid proteins has been shown to contribute to CTL-mediated clearance of Ad-transduced cells in addition to stimulating B cells to produce neutralizing antibodies [59-60].

Activation of T cells by antigens requires a complex program of molecular interactions between the T cell and an APC, each of which could be a target for immune blockade in gene therapy. Initiation of a T-cell-mediated response requires APCs which present short peptides derived from ingested foreign antigens (e.g., a virus particle) in association with major histocompatability (MHC class II) molecules to interact with the T-cell receptor (Fig. 2a). In addition, there are other signals that are needed for successful stimulation of the T-cell response. These include B7-1 (CD80) and B7-2 (CD86) ligands present on antigen presenting cells which bind to the CD28 / CTLA4 receptors on T cells and elicit a costimulatory response needed for this activation [61]. Such costimulatory responses result in cytokine secretion and full T-cell activation (Fig. 2a). There are two functionally distinct subclasses of helper T cells that can be distinguished by the interleukins that they secrete upon activation. Thl cells secrete IL-2 and gammainterferon and are concerned mainly with helping cytotoxic T cells and macrophages, while Th2 cells secrete interleukin (IL)-4 and IL-5 and are concerned mainly with helping B cells (Fig. 2a).

Once activated, the helper T cell can stimulate a B cell that specifically displays the same complex of foreign antigen and MHC class II protein on its surface (Fig. 2b). T-cell dependent activation of B cells in turn leads to upregulation of CD40 ligand (CD40L) on the T cell, promoting the engagement of CD40 on the cognate B cell [62] (Fig. 2b).

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