140. Primary Adenovirus Specific Cytotoxic T Lymphocyte Response Occurs after Viral Clearance and Liver Enzyme Elevation

Abstract

Top of pageAbstract The relative contribution of the specific cytotoxic T lymphocyte (CTL) response and innate immune response to the clearance of adenovirus (Ad) and its transgene in the context of gene therapy has been a subject of debate. Transient transgene expression was believed due to the elimination of Ad transduced cells by CTLs. To determine the relative contribution of specific CTL killing of Ad-infected hepatocytes, we have utilized a wild-type Ad5 to study viral clearance from the liver and its associated liver enzyme elevation during a primary immune response. Human Ad5 replicates poorly in mice due to species restriction but induces a dominant CTL response to the E1B peptide (AA192 VNIRNCCYI) associated with mouse class I MHC antigen, H2-Db, in C57BL/6 (B6) mice. Thus, Ad5 was used to mimic a low replicative Ad vectors used in humans and an MHC-I tetramer that contains the E1Bp, H2-Db and b2-microglubulin (Db-E1B), was developed and used to detect the Ad-specific CTLs in Ad5-primed mice. After Ad5 infection, the Db-E1B+CD8+T cells were undetectable in the spleen, liver and lung until day 5, and peaked at day 8. At the peak of the CTL response, 8 days post infection, 10% of CD8+ cells in the spleen. Surprisingly, 45% in the liver and 55% in the lung of the CD8+ T cells were Db-E1B+, indicating that most CD8+ T cells in target tissue are specifically recruited to these sites. Furthermore, nearly 100% of Db-E1B+CD8+ cells were IFN|[gamma]| and granzyme-B positive with intracellular staining, indicating that the recruited CD8+CTL exhibit potent killing activity. The E1B-specific in vivo CTL response was detected by the transfer of CFSE-labeled, E1B192 peptide-pulsed target cells from H-2b mice into Ad5-primed syngeneic mice. The magnitude of the in vivo CTL response correlated with the number of Db-E1B+ CTLs in the spleen and liver (r2=0.96, p<0.01). Interestingly, viral DNA genome and E1B mRNA peaked at day 2 and reduced rapidly by day 6 and then maintained at a stable level without significant change 200 days after infection. Viral clearance correlated with the peak liver enzyme response that occurred on day 4, before CTLs became detectable. In contrast, there was no further change in the level of viral DNA genome and E1B mRNA in the liver or lung as a result of the peak CTL response at day 8. Administration of Ad5 to athymic SCID mice induced no CD8 T cells or in vivo specific CTL response against E1B192 peptide, yet viral clearance and liver enzyme response in SCID mice were comparable to those of B6 mice. Taken together, our results suggest that the innate response, but not the CTL response, plays a dominant role in elimination of Ad during a primary response. Stable long-term expression of lower levels of virus and transgene persist for 200 days despite the presence of potent CD8+ CTLs, indicating that they reside in liver cells that are resistant to CTL elimination. These findings will be useful in developing efficient and safe Ad gene therapy protocols.