(a) Schematic time line representation of two administrations of a first-generation … Two doses of AdCMVHSV1-tk were given i.v. 4 weeks apart to three macaques. Only two of the animals received immunosuppression. PET imaging was performed 2 days after each adenovirus administration. The liver of the three animals showed evidence of transgene expression following the first administration, but Vorinostat MK0683 only one of the immunosuppressed subjects showed a certain degree of reexpression upon the second administration (Figure 1b,c). To rule out residual transgene expression from the first dose of adenovirus, a PET study was performed 1 week before the second adenoviral administration. This is shown in Supplementary Figure S1a that summarizes the sequential PET measurements of transgene expression performed in these animals.

In the nonhuman primates, the two-drug regime reduced the titer of neutralizing antibodies by one log, but failed to abolish the humoral response (Figure 1d). One potential reason is that the depletion of B cells had not been complete as previously suggested by other authors.22 In this regard, we found that in the subject who attained HSV1-tk partial reexpression, CD20+ B-cells in peripheral blood samples were virtually undetectable (Figure 1e). In the other animals treated with the same immunosuppressive regimen, B-cell counts were lowered only about 2.5 times in comparison to the control macaque (Figure 1e). This difference could account for the different gene-transfer outcome.

Peripheral blood T-lymphocytes (both CD4 and CD8) proliferated avidly upon in vitro exposure to adenoviral capsids, 6 weeks after the first administration (Figure 1f,g). Clearly less marked, albeit detectable, proliferation was substantiated in the CD4 T-cell compartment of the animal with partial transgene reexpression, and it is likely that these T-lymphocytes were still providing sufficient help for antibody responses (Figure 1f,g). Of note, all the animals tolerated the treatments well with only moderate increases of serum liver enzymes (Supplementary Figure S1b). More intensive immunosuppression with Rituximab, FK506, antithymocyte immunoglobulin, MMF, and steroids permit repeated transfer of the HSV1-tk transgene The partial success obtained in our first study (Figure 1) suggest that B-22 and T-cell suppression is important for permitting adenoviral vector readministration, but that the two-drug regimen may not be sufficient.

1 Therefore, we pursued a more intensive immunosuppressive protocol. Accordingly, a more intense five-drug immunosuppression regime was tested in a new group of three animals. Antithymocyte immunoglobulin (ATG),19,27 mycophenolate mofetil (MMF),28 and steroids were added. The course of Rituximab27 was intensified with weekly doses between adenoviral administrations. Batimastat FK506 was given as in the first group of macaques (Figure 2a).