Steroids were included mainly to prevent systemic inflammatory responses elicited by ATG and in addition methyl-prednisolone can also diminish the early innate immune response2 toward the adenoviral capsids. Figure 2 An uninterrupted five-drug immunosuppressive regimen (Rituximab+FK506+MMF+ATG+methyl sellectchem prednisolone) permits efficient liver gene-transduction upon a second administration of an adenoviral vector given 1 month later to an … Preexisting low titers of antiadenovirus antibodies seen in patients had not precluded gene transfer upon first intratumoral administrations of the vector.15 Such weak reactivity probably reflects previous exposure to partially cross-reactive adenoviral serotypes. None of the animals in the first group had shown this feature (data not shown).

However, one of the macaques in the second group showed a low titer of pretreatment antibodies that fell below the detection threshold in four other sequential samples (Figure 2b). In this subject, a weak cellular response toward adenoviral capsids was also detected immediately before AdCMVHSV1-tk exposure (Figure 2c). None of the other two animals showed pretreatment signs of antiadenoviral immunity (Figure 2b,c). To study the possible influence of this weak pretreatment antiadenoviral immunity, this animal was chosen to receive immunosuppression, whereas the other two were randomly distributed to receive or not the five-drug regimen. Again all the macaques showed intense PET signal following the first adenovirus exposure (Figure 2d).

Upon second administration 1 month later (Figure 2d), one of the immunosuppressed macaques remarkably showed liver transgene expression of the same order of magnitude as in the previous administration. As expected, the control subject did not show reexpression of the transgene. This was also the case with the animal which had showed low levels of pretreatment antiadenoviral immunity. In the animal with Brefeldin_A positive PET signal upon adenoviral readministration, immunohistological (Figure 2e) and immunoblot analyses (Figure 2f) on ultrasound-guided needle biopsies taken from the right and left liver lobes of the animals confirmed the expression of the tk transgene. There were no histological signs of liver inflammation although serum transaminases were moderately increased (Supplementary Figure S2a,d). Tk was present both in parenchymal cells and liver macrophages (Kupffer cells) (Figure 2e and Supplementary Figures S2b,c and S4). Kupffer cells were identified by morphological criteria and CD68 immunostaining in serial sections (Supplementary Figure S2c). Quantitative analyses 3 days after the adenoviral readministration concluded that about 8% of hepatocytes and virtually all Kupffer cells were HSV1-tk+ (Figure 2e).