1090. Adenovirus Fiber Incorporates an Immunodominant MHC Class I Restricted Epitope Peptide

Abstract

Top of pageAbstract Adenovirus has been extensively investigated as one of the most promising gene delivery vectors. One of the major limitations to its application is the generation of a strong CD8+ cytotoxic T lymphocyte (CTL) response. However, analysis of the viral specific cytotoxic T lymphocyte (CTL) response and the correlation with the elimination of vector and transgene has not been possible due to the lack of a specific method to detect viral antigen-induced CTLs. To identify Adenovirus specific CTLs generated after administration of recombinant adenovirus vectors, we utilized two MHC binding peptide prediction programs, BIMAS and SYFPEITHI, to predict epitope peptides from adenovirus hexon, penton and fiber proteins that are processed and presented by the mouse MHC class I H2-Kb antigen. To test the specificity of the predicted peptides, the specific CTL activity was determined by an in vivo CTL assay and an IFN|[gamma]| ELISPOT assay. C57BL/6 (B6; H2b) mice were i.v. injected with 8|[times]|108i.u of wild type Ad5 through the tail vein. Eight days later, spleen cells from naive B6 mice were labeled with high (2uM; CFSEHi) concentration of CFSE and then pulsed with predicated peptides, respectively, before co-transferred to the wild type Ad5 primed B6 mice (recipient) with identical number of unpulsed CFSELo (0.2uM) cells for in vivo CTL assay. The known immunodominant E1B peptide (AA192|[ndash]|200 VNIRNCCYI ) of Ad was used as a positive control and human flu M1 peptide (AA58|[ndash]|66 GILGFVFTL) was used as a negative control. After six hours, spleen cells and liver mononuclear cells (MNCs) were recovered from recipient mice and analyzed by flow cytometry and by the IFN|[gamma]| ELISPOT assay. In the spleen of immunized B6 mice, 85.9|[plusmn]|13% of fiber peptide (AA391|[ndash]|399 VGNKNNDKL) pulsed CFSEHi target cells were specifically lysed, compared to 98.2|[plusmn]|5% of E1B positive control peptide pulsed cells and no specific lysis to M1 negative control peptide pulsed cells. There were 202|[plusmn]|18 spot forming cells (SFC) per 106 liver MNCs in immunized mice that were responsive to fiber peptide (AA391|[ndash]|399 VGNKNNDKL) and 468|[plusmn]|87 SFC/106 liver MNCs specific for E1B peptide, compared to 10|[plusmn]|10 SFC/106 liver MNCs specific for M1 peptide. There were no SFCs specific for other predicted peptides and no specific killing in target cells pulsed with other peptides (data not shown). To further confirm the immunogenicity of the fiber peptide in the context of E1-deleted Ad vectors, B6 mice were injected with 109i.u of AdLacZ and then analyzed by ELISPOT assay. There were 109|[plusmn]|23/106 liver MNCs specific this fiber peptide, whereas no SFCs specific for E1B peptide or M1 peptide. These results indicated that the fiber peptide is a strong immunogenic peptide and the in vivo CTL assay could be used as a sensitive method to measure Ad-specific CTL response to any viral vectors that derived from Ad2 or Ad5 virus. These findings will provide us with a powerful tool for assessing immune response to Ad capsid in the context of gene therapy.