Immune response to recombinant adenovirus in humans: capsid components from viral input are targets for vector-specific cytotoxic T lymphocytes

Abstract

We previously demonstrated that a single injection of 10(9) PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218-2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8(+) CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses.

FIG. 1

(A) Northern blot analysis of LCL infected with first- and second-generation vectors. LCL were infected with 20,000 particles of AdE1° (E1 deleted), AdE1E2° (E1 and E2 deleted), or AdE1E4° (E1 and E4 deleted) per cell and 10 PFU of Vac-Fi (recombinant vaccinia virus encoding the adenovirus type 5 fiber) per cell as previously described (9). 293 cells infected with wild-type adenovirus were used as positive controls. All these viruses were produced by Transgene SA. Adenovirus vector construction was described in reference . Total RNA was extracted 60 h postinfection for adenovirus vectors and 24 h for Vac-Fi (the delay was used to obtain maximal transcription). RNA (10 μg) was separated on a formaldehyde-agarose gel, transferred to a nitrocellulose membrane, and probed for the fiber with a ³²P-labeled fragment of the fiber sequence. (B) CTL recognition of first- and second-generation adenovirus vectors. CTL were assayed in the presence of LCL infected 40 h prior to the test with 20,000 particles of AdE1° or AdE1E2° per cell, or mock infected, in a standard 4-h chromium release assay.

FIG. 2

Effect of the viral gene transcription block on CTL lysis. (A) LCL treated with Act-D (5 μg/ml) 30 min prior to and during infection or left untreated were infected for 1 h with 12,000 particles of Ad-CFTR per cell and then resuspended in 5 ml of complete medium with or without 0.2 μg of Act-D per ml. After 24 h of culture, CTL recognition was assayed on autologous mock- or adenovirus-infected Act-D-treated and untreated targets in a standard 4-h chromium release assay (a concentration of 0.2 μg of Act-D per ml was maintained throughout the test). (B) In parallel, adenovirus-infected ⁵¹Cr-labeled targets were seeded in triplicate in 96-well plates (1.5 × 10⁵ cells/well) and RNA transcription was measured by [³H]uridine (1 μCi/well) incorporation after 18 h of culture. E:T, effector-to-target-cell ratio.

FIG. 3

Effect of blockage of viral peptide presentation on CTL lysis. LCL treated with BFA (1 μg/ml 30 mn prior to and during infection) or left untreated were infected for 1 h with 1,000 particles of Ad-CFTR per cell. After 18 h of culture, CTL recognition was assayed on autologous mock- or adenovirus-infected, BFA-treated and untreated targets, in a standard 4-h chromium release assay (a concentration of 1 μg of BFA per ml was maintained throughout the test). E:T, effector-to-target-cell ratio.

FIG. 4

Patient 1 (A) and patient 2 (B) CTL recognition of capsid components. CTLs were tested for 10 min at 37°C by an ELISPOT assay for the secretion of IFN-γ in the presence of autologous LCL (5,000 cells/well) loaded using a hypertonic solution (0.5 M sucrose, 10% polyethylene glycol 1,000, and 10 mM HEPES in RPMI 1640) with 20 μl of recombinant capsid components (penton base, hexon, and fiber) or control Sf9 cell extract (Sf9 plus 500 μl of hypertonic solution). Adenovirus-infected targets (12,000 particles of Ad-CFTR per cell) were used as positive controls, and the results are expressed as the number of specific spots per well after subtracting the background determined with uninfected unloaded LCL.