The humoral and cellular immune response to recombinant adenoviral vectors, as described in several animal models, result in extinction of transgene expression, severe local inflammation, and production of neutralizing antibodies that prevent readministration [59, 63, 64]. A direct correlation between neutralizing antibody and the block to readministration of vector has been established by passive transfer of immunity by sera from treated to naive animals , One approach to enhance adenoviral-mediated gene transfer is to modulate the host immune response by immunosuppression of the recipient organism.
Chronic immune suppression with drugs such as cyclosporine and cyclophosphamide has improved the stability of adenovirus-encoded transgene expression in animal models of liver-, lung-, and muscle-directed gene therapy [64-66]. Cyclophosphamide is a commonly used immune suppressive agent for the treatment of autoimmune diseases and prevention of rejection following allograft organ transplantation . It is activated by hepatic cytochrome p450 to metabolites that exhibit toxicity primarily to dividing cells, including activated T and B cells .
Administration of cyclophosphamide with intravenous infusion of adenoviral vector blocked activation of both CTL and T helper cells, resulting in prolonged transgene expression in the liver with reduced anti-Ad neutralizing antibody production , A similar effect was seen in the lung; however, a much lower dose of cyclophosphamide was needed to prevent neutralizing antibody formation. In contrast, stabilization of transgene expression was achieved only at a high dose. This difference may be a consequence of differences in the route of administration of the vector, which could result in differences in presentation of antigens. For example the intravenous route more likely deposits larger quantities of antigens to tissue enriched with antigen-presenting cells such as the spleen. In addition neutralization of virus in the lung is restricted to the Th2-dependent isotope, which is easier to ablate than ablation of both Thl and Th2 subsets which contribute to formation of antiviral responses when vector is delivered systemically .
In contrast cyclosporin (CSA) alone failed to reduce the production of neutralizing antibodies to cFIX in hemophilia B dogs but was effective at prolonging gene expression of FIX [64, 65]. CSA reportedly inhibits early events in T-cell activation such as activation of interleukin-2 gene expression , which may explain why CSA most likely affected the cellular rather than the humoral immune response following adenovirus-mediated gene therapy in the hemophilia B dogs.
One of the main concerns with the protocols used in animal models for general immunosuppression is the high dose necessary to successfully obtain readministration of gene therapy vectors. This is substantially higher than approved doses for use in humans. Thus it remains to be established whether clinically acceptable doses (presumably lower doses) may indeed have the same effect on immunosuppression and allow readministration of gene therapy vectors in a clinical setting.
Bouvet et al.  report that etoposide at clinically acceptable doses suppresses the formation of neutralizing antibodies and CTLs to adenovirus and results in successful intratumor transgene expression in immunized mice. Etoposide is a semisynthetic derivative of podophyllotoxin that causes an arrest at G2 of the cell cycle. It inhibits DNA synthesis by interfering with the enzyme topoisomerase II and leads to cell death by apoptosis . Thus repeated adenoviral-mediated gene therapy may be achievable in cancer patients who are concurrently undergoing treatment with chemotherapy.
Most of the immunosupressants discussed so far have the distinct disadvantage of causing general immunosuppression that may not be desirable in some clinical settings. An alternative immunosuppressant, deoxyspergualin, (DSG), with more selective properties has been shown to be useful in readministration of systemically delivered viral vectors expressing Factor IX  or lung-directed viral vectors expressing the human cystic fibrosis conductance regulator (hCFTR) . Deoxyspergualin interferes with the differentiation of B and T cells and also with antigen processing. An important property of DSG is that it does not induce a general suppression of the immune system, but rather results in a selective lack of response to specific antigens presented at the time of drug treatment.
The central role of the CD4+ T cell provides a strategy to prevent humoral and cellular responses to adenovirus vectors through a transient blockade of CD4+ T-cell activation at the time of vector administration. The rationale for this approach is that chronic immune suppression should not be necessary if the primary stimulus for activation is the input capsid proteins. In support of this hypothesis, it has been shown that depletion of CD4+ T cells with a monoclonal antibody (GK1.5) at the time of vector administration can effectively prevent CTL and B-cell responses in murine models of liver- and lung-directed gene therapy [74-76].
Selective inhibition of the TH2 subset of T helper cells by administration of the cytokine interleukin 12 or gamma interferon (IFNy) with adenovirus vector has prevented the humoral immune response in mouse lung tissue . The success of this approach, however, depends on the relative contribution that Th2-dependent immunoglobulin (Ig) isotypes play in virus neutralization, the profile of which may be affected by strain and species of animal as well as routes of vector administrations. Th2-specific ablation with IL-12 is an effective approach for lung-directed gene therapies in the mouse where IgA is the primary source of neutralizing antibodies. However, in the case of the mouse liver both Thl and Th2 cells contribute to the production of virus-specific antibodies and although IL-12 reduced the total amount of neutralizing antibody in this organ it was not enough to allow effective readministration of the virus .
Interfering with the distal pathway of CD4+ activation by administering CTLA4 immunoglobulin (Ig) with adenovirus vector improved the stability of recombinant gene expression in mouse liver but did not significantly impact neutralizing antibody production or allow systemic vector readministration . muCTLA4Ig is a chimeric protein of murine immunoglobulin IgG2a fused to murine CTLA4 and is an inhibitor of the CD28-B7 pathway (Fig. 2a). In contrast in the lung huCTLA4-Ig treatment significantly blocked the formation of neutralizing antibodies allowing efficient readministration of virus, whereas transgene expression was only moderately prolonged .
Differences between the effects of this drug on humoral and cellular responses in lung versus liver are surprising. It is known that secretory IgA, which depends on Th2 cells, contributes to neutralization in the lung, whereas antibodies of other isotypes, such as IgGl and IgG2a, neutralize virus administered systemically. As previously discussed, different routes of virus instillation may result in different mechanisms of antigen presentation, which could affect inhibition of B- and T-cell activation by CTLA4Ig. For example, local injection of an adenovirus vector expressing CTLA4 Ig into the brain suppressed not only local cell infiltration in this tissue but also reduced the humoral immune response to adenovirus .
The expression of CD40L by activated helper T cells (Th) triggers B cell cycling through binding to CD40 (Fig. 2b). CD40L is expressed transiently at high levels on activated CD4+ T cells [81, 82], The costimulation provided by CD40L with CD40 is essential for thymus-dependent humoral immunity [82, 83] and is also thought to play an important role in the generation of cellular immune responses through the production of helper cytokines [83, 84]. A transient block of costimulation between T cells and B cells and other antigen-presenting cells using a monoclonal antibody against CD40 ligand (MR-1) suppressed the development of antibodies against Ad delivered to mouse or nonhuman primate lung, in addition to decreasing the cellular immune response to the vector [85-87], This in turn resulted in an increase in persistence of transgene expression. Furthermore, when MR1 was administered with a second dose of Ad vector to mice preimmunized against vector, it was able to interfere with the development of a secondary antibody response and allowed for high levels of transgene expression upon a third administration of vector to the mouse airway , Similarly in the nonhuman primate lung administration of a humanized anti-CD40 ligand MAb (hu5C8) at the time of vector instillation, markedly suppressed adenovirus-induced lymphoproliferation and cytokine responses, in addition there was a marked suppression of IgA and neutralizing antibodies which permitted vector readministration ,
In the case of systemic delivery of vector it is thought that only CD40 ligand blockade inhibits anti-Ad antibody generation sufficiently to allow redosing to the liver . Thus a combination of anti-CD40 ligand and murine CTLA4Ig was necessary to allow transduction after secondary vector administration in mouse liver, whereas neither agent alone was sufficient .
Orally administered antigens have been shown to induce systemic unresponsiveness to a subsequent exposure to the antigen. Oral tolerance primarily results in active suppression by regulatory T cells, clonal anergy, or clonal deletion. What activates one mechanism over the other is not altogether clear, although it is generally agreed that lower doses of antigen more likely lead to suppression and higher doses to clonal anergy or deletion . In addition, the form of antigen and frequency of feeding influences the type of tolerance induced .
Long-term adenovirus-based gene expression was observed in the liver of rats with preexisting anti-adenovirus antibodies that were tolerized by feeding viral proteins (11 x 1-mg dose) . In the tolerized rats the anti-adenovirus humoral immune response was downregulated allowing systemic readministration of vector. Moreover, in the tolerized rats vector readministration did not lead to a secondary humoral immune response, suggesting that repeated adenovirus-directed gene transfer may be possible despite the presence of a residual antibody titer from a previous exposure to vector ,
Similarly, this approach has been used to enhance gene transfer to the rat parotid gland which is within the mucosal immune system . As it is more difficult to induce complete mucosal tolerance compared to systemic tolerance only partial tolerance to mucosally applied viral vectors was achieved. A different feeding regimen of adenovirus (5 x 50 (xg dose) along with a different form of antigen, UV inactivated vector, in addition to a difference in route of viral challenge may explain the differences in the results from the two studies. Nonetheless, it is possible to induce some degree of tolerance to mucosally applied adenovirus by feeding animals the virus. In clinical situations, some concern may exist about tolerizing a host toward adenoviruses, which can be pathogenic in humans.
One strategy to circumvent Ad vector-specific neutralizing immunity is to switch the serotype of the Ad vector [94, 95]. As already discussed in section II.A, there are 51 serotypes that are classified on the basis of biological, chemical, immunological, and structural properties into six subgroups and then into serotypes based on neutralization by antisera to other Ad serotypes. Following an initial administration of adenovirus, serotype-specific antibodies are generated against the major viral capsid proteins (section II.B). Group C adenoviruses include Ad2 and Ad5, the more commonly used serotypes for gene therapy vectors. While the capsid proteins of the group C adenoviruses are highly conserved, viruses from a different subgroup have capsid proteins which are only weakly homologous to the group C viral capsid proteins. Thus the immune response against the non-group-C viruses in many instances does not block infection by a group C virus . This was demonstrated in a study where Sprague-Dawley rats were injected intraperitoneally (ip) with wild-type Group B virus, WT Ad7, either alone or sequentially with WTAd4 (group E) prior to intracardial administration of an Ad5-based gene transfer vector . Transgene expression in all animals that received non-group-C viruses prior to Ad5 was equivalent to naive animals. In contrast, animals that received WT Ad5 prior to the Ad5-based gene transfer vector had greatly reduced levels of transgene expression compared to naive animals.
Similarly, in the development of gene therapy vectors for the lung and the treatment of cystic fibrosis, it was shown that intratracheal administration of an immunizing does of wild-type Ad 4 (subgroup E) or Ad30 (subgroup D)
did not affect the subsequent expression of human CFTR from an Ad5 based gene transfer vector . More importantly the alternate use of Ad vectors from different serotypes (Ad2, Ad5) within the same subgroup (C) can also circumvent anti-Ad humoral immunity and permit effective gene transfer to the lung  and to the liver  upon repeat administration. In the context of future clinical applications for this approach it is relevant that Ad2- and Ad5-based vectors can be administered alternately as these are the Ad serotypes that are in current use in human clinical trials.
Alternative approaches have been developed for circumventing antibody neutralization of Ad vectors that are centered on modification of the Ad virion rather than on immunosuppressive treatment of recipient animals [98-101]. One such approach involves the covalent attachment of the polymer PEG (polyethylene glycol) to the surface of adenovirus [99, 100]. Covalent modification of Ad virions using chemically reactive PEG renders the virus less susceptible to neutralization, due to shielding of neutralizing epitopes on the surface of the virus by PEG molecules. The components of the capsid that elicit a neutralizing immune response, i.e., hexon, fibre, and penton base (see section II.B) are the main targets for PEGylation. Importantly the covalent attachment of a PEG polymer to the surface of the adenovirus can be achieved with retention of infectivity, while PEG-modified adenovirus have been shown to be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus [99, 100].
Similarly, Beer et al.  demonstrated that adenovirus vectors could be formulated in a polymer preparation of PLGA (poly(lactic/glycolic acid) with retention of bioactivity. Mice immunized subcutaneously with encapsulated recombinant adenoviral vectors show a greater than 45-fold reduction in anti-adenovirus titers relative to nonencapsulated vectors. Although the authors do not show any in vivo data they postulate that the process of encapsulation of a vector in a polymer preparation may potentially mask the adenovirus from circulating antibodies. This is also based on the observation that encapsulated vectors are less susceptible to neutralization than nonencapsulated vectors in vitro. The possible disadvantages of this approach include the efficiency at which the Ad vector is released from the polymer and the true demonstration that this approach has any advantage in vivo.
More recently another nongenetic strategy to modify the surface of the virion has been described. This involves a covalent coating using a multivalent hydrophilic polymer based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Multivalent polymeric modification of adenovirus rendered the virus less susceptible to neutralization by anti-adenovirus antibodies. As with the studies with PLGA this approach was shown only to be effective in vitro and may not be applicable in an in vivo setting.
Another novel approach to overcoming the problem of serum neutralizing antibodies was described by Chen et al. , and involves the principle of immunoapheresis. An affinity column consisting of cloned recombinant capsid proteins was generated to specifically remove anti-adenovirus antibodies from human clinical serum samples. The authors postulate that such an affinity column could be used in conjunction with apheresis in a technique called immunoapheresis. During the apheresis procedure, patients' serum could pass through this immunoaffinity column removing anti-adenovirus antibodies. Anti-adenovirus antibodies would be expected to repopulate the vascular compartment eventually but a temporal window of several hours for intravascular adenovirus therapy could be created ,
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