Generation of Cytotoxic T Lymphocytes against ... - Journal of Virology

JOURNAL OF VIROLOGY, Mar. 1997, p. 2277–2284 0022-538X/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 71, No. 3

Generation of Cytotoxic T Lymphocytes against Immunorecessive Epitopes after Multiple Immunizations with Adenovirus Vectors Is Dependent on Haplotype TIM E. SPARER,1 SUSAN G. WYNN,1 DANIEL J. CLARK,1 JOHANNE M. KAPLAN,2 LISA M. CARDOZA,2 SAMUEL C. WADSWORTH,2 ALAN E. SMITH,2 AND LINDA R. GOODING1* Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322,1 and Genzyme Corporation, Framingham, Massachusetts 017012 Received 1 May 1996/Accepted 9 December 1996

Currently, adenovirus (Ad) is being considered as a vector for the treatment of cystic fibrosis as well as other diseases. However, the cytotoxic T lymphocyte (CTL) response to Ad could limit the effectiveness of such approaches. Since the CTL response to virus infection is often focused on one or a few immunodominant epitopes, one approach to circumvent this response is to create vectors that lack these immunodominant epitopes. The effectiveness of this approach was tested by immunizing mice with human group C adenoviruses. Three mouse strains (C57BL/10SnJ [H-2b], C3HeB/FeJ [H-2k], and BALB/cByJ [H-2d]) were immunized with wild-type Ad or Ad vectors lacking the immunodominant antigen(s), and the CTL responses were measured. In C57BL/10 (B10) mice, a single inoculation intraperitoneally (i.p.) led to the recognition of an immunodominant antigen in E1A. When B10 mice were inoculated multiple times either i.p. or intranasally with wild-type Ad or an Ad vector lacking most of the E1 region, subdominant epitopes outside this region were recognized. In contrast, C3H mice inoculated with wild-type Ad recognized an epitope mapping within E1B. When inoculated twice with Ad vectors lacking both E1A and E1B, no immunorecessive epitopes were recognized. The immune response to Ad in BALB/c mice was more complex. CTLs from BALB/c mice inoculated i.p. with wild-type Ad recognized E1B in the context of the major histocompatibility complex (MHC) class I Dd allele and a region outside E1 associated with the Kd allele. When BALB/c mice were inoculated with E1-deleted Ad vectors, only the immunodominant Kd-restricted epitope was recognized, and Dd-restricted CTLs did not develop. This report indicates that the emergence of CTLs against immunorecessive epitopes following multiple administrations of Ad vectors lacking immunodominant antigens is dependent on haplotype and could present an obstacle to gene therapy in an MHC-diverse human population. CD81 cytotoxic T lymphocytes (CTL) are important for viral clearance in many virus systems (for a review, see reference 8). CTL recognize major histocompatibility complex (MHC) class I molecules bound to peptides of 8 to 10 residues in length derived from endogenously synthesized proteins (36). In spite of the hundreds of potential antigens that could be recognized by class I-restricted CTL during a viral infection, the majority of the immune response is focused on only one or a few immunodominant antigens. For example, even though influenza virus and human immunodeficiency virus produce complex proteins such as hemagglutinin or gp160 with multiple potential CTL epitopes, the CTL response is dominated by a few immunodominant epitopes (3, 33). Similarly, the mouse CTL responses to the 708-amino-acid T antigen of simian virus 40 (SV40) are focused on one to three epitopes that vary with the major histocompatibility complex (MHC) class I allele (28, 34). Recently, the immune response to adenovirus (Ad) has become increasingly important because Ad is being examined as a vector for in vivo gene therapy. Genes inserted into E1deficient (DE1A and E1B) Ads are being tested for various gene therapy applications, including replacement of defective tumor suppressor genes in some cancers (6), the low density lipoprotein receptor gene in familial hypercholesterolemia patients (17), and the cystic fibrosis transmembrane conductance

regulator (CFTR) gene in cystic fibrosis patients (43). Although successful Ad-mediated gene transfer of CFTR has been reported both in vitro and in vivo (37, 43, 44) immunological responses to Ad vectors may limit their usefulness. Yang et al. (39, 40) reported that the CTL response to Ad contributes to the elimination of transgene expression in vivo. CTL recognizing viral proteins could eliminate the cells expressing the transgene product, thereby requiring readministration of the Ad vector. If this is the case, then preventing recognition of the Ad vector by CTL would allow for longer intervals between vector readministrations. Thus, an understanding of the CTL response to Ad might provide useful information for the future design of Ad vectors. Previous studies have revealed that when CTL are generated against wild-type Ad, they recognize E1A as the immunodominant antigen in B10 mice but not in C3H or BALB/c mice (27). In this report, we set out to determine whether multiple immunizations altered the CTL response and whether the CTL response differed after intranasal (i.n.) versus intraperitoneal (i.p.) inoculations, as well as to identify the immunodominant antigens recognized by C3H and BALB/c mice. MATERIALS AND METHODS Cells and viruses. SV40-transformed fibroblasts (SVB6KHA from C57/BL6 mice, SV2R from B10.A (2R) mice, PSC3H from C3HeJ mice, SVBALB from BALB/c mice, SVHTI from HTI mice, and SVHTG from HTG mice as described elsewhere (3, 14, 29]) were grown in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, N.Y.) supplemented with 10% fetal calf serum (Hyclone, Logan, Utah) and 1% L-glutamine (GIBCO). Viruses were grown on KB cells and plaqued on A549 cells (American Type Culture Collection [ATCC],

* Corresponding Address: Department of Microbiology and Immunology, Emory University School of Medicine, 3107 Rollins Research Building, 1510 Clifton Rd., Atlanta, GA 30322. 2277

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J. VIROL. with 200 mCi of Na51 2 CrO4 (New England Nuclear, Boston, Mass.) on the day prior to assaying. CTL activity was measured by a 5-h 51Cr release assay. Results were calculated from the mean of triplicate samples and expressed as (E 2 C)/(M 2 C) 3 100 5 percent specific lysis, where C equals counts per minute released by nonstimulated lymphocytes, E equals counts per minute released by sensitized lymphocytes, and M equals maximum releasable counts per minute, determined by the addition of 0.05 ml of 1 N HCl to 0.05 ml of target cells. Data are percent specific lysis 6 standard errors of the means. The graphs presented are representative of three to five experiments with at least two mice per group in each. Detection of infectivity. To confirm that the target cells were infected, 5 3 103 target cells were allowed to adhere to a multispot microscope slide (Shandon, Pittsburgh, Pa.) for 5 h. The cells were then fixed in methanol (2208C) for 10 min and treated with the DAKO LSAB (R) II kit (DAKO Corp., Carpinteria, Calif.) by using a rabbit anti-72K (DNA binding protein) serum (a gift of Daniel F. Kessling) as the primary antibody. Briefly, endogenous peroxidase was neutralized with 3% H2O2 for 5 min. The cells were then incubated with the anti-72K serum (1:500 in phosphate-buffered saline [PBS] plus 1% fetal calf serum) for 30 min at room temperature (RT). The slides were washed three times with PBS and incubated with biotinylated anti-rabbit antibody for 30 min at RT. The slides were again washed three times with PBS and subsequently incubated with streptavidin peroxidase for 30 min at RT. The cells were then washed five times with PBS and incubated with 3% 3-amino-9-ethylcarbazole for 10 min at RT. The slides were washed in H2O, covered in 90% glycerol, and examined under a light microscope for infectivity. In the experiments described here, greater than 85% of the target cells were infected.

FIG. 1. E1A/E1B mutants used in this study. The Ad nucleotide map of E1A/E1B is shown at the top. Open boxes, proteins; filled boxes, deletions. The nucleotides deleted in each mutant are noted. The E1A/E1B mutants are dl312 (D448-1349), dl313 (D1334-3639) (18), Ad2/CFTR-2 (D357-3328) (2), and Ad2/ CFTR-7 (D545-4020) (this study).

Rockville, Md.), except for viruses lacking E1A and/or E1B, which were grown and plaqued on 293 cells (ATCC) as previously described (16). The virus mutants used in this study are summarized in Fig 1. dl801 (4) is an Ad type 2 (Ad2)-based mutant that lacks most of E3 but retains 14.7K. dl309 (18) is an Ad5 virus lacking the E3 proteins 10.4K, 14.5K, and 14.7K but maintaining E3 gp19K. dl327 (35) is deleted for almost the entire E3 region including gp19K. dl754 (15) lacks only gp19K and part of 6.7K. Ad2/CFTR-2 is a recombinant Ad2 vector in which most of the E1 region (nucleotides 357 to 3328) has been replaced with CFTR cDNA flanked by the phosphoglycerate kinase promoter and a bovine growth hormone polyadenylation site. The E3 region is conserved, while the E4 region is modified by removal of all open reading frames (between nucleotides 32815 and 35577) and replacement with the E4 open reading frame 6 (nucleotides 33178 to 34082). Ad2/CFTR-7 is a similar construct, in which the entire E1 region including the protein IX gene (nucleotides 546 to 4020) was replaced with an expression cassette encoding the E1A promoter, CFTR cDNA, and the early polyadenylation site from SV40. Ad2/ CFTR-7 contains the same E4 modification as Ad2/CFTR-2, with an additional deletion in the E3 region (nucleotides 29293 to 30840; gp19K retained). Since Ad5 and Ad2 are 99 to 100% identical at the nucleotide level and have been shown to be completely cross-reactive for CTL, dl801, dl309, Ad5, and Ad2 were used interchangeably as positive controls for lysis (23, 29). Mouse infections. Six- to eight-week-old male C57BL/10SnJ, BALB/cByJ, and C3HeB/FeJ mice (Jackson Laboratories, Bar Harbor, Maine) were inoculated intraperitoneally (i.p.) with 2 3 107 PFU of Ad5. For intranasal (i.n.) inoculations, mice were anesthetized with Avertin (2,2,2-tribromoethanol) (Aldrich Chemical Co., Milwaukee, Wis.), and 25 ml of 1011 PFU of CsCl-banded virus per ml was instilled i.n. (31). For double immunizations, a second administration was given 7 days following the first inoculation. Spleens were harvested 7 or more days following the final inoculation. CTL assays. The spleens from i.n. or i.p. inoculated mice were removed, stimulated in vitro for 6 days with syngeneic, Ad-infected g-irradiated stimulators, and assayed against 51Cr-labelled targets as previously described (27, 29). Briefly, for Db-restricted CTL, splenocytes were stimulated in vitro with SVB6KHA (KbDb) fibroblasts infected with dl754 or dl801 and assayed against virally infected SV2R targets (Kk Db) or the E1A transfectant, L13–14, which has been described elsewhere (27). SV2R was chosen as the target cell because E3 gp19K does not suppress CTL recognition in this cell line (26a). For Kk-restricted CTL, splenocytes were restimulated in vitro with PSC3H (Kk Dk) fibroblasts infected with either dl754 or dl801 and were assayed against PSC3H targets. For Kd and Dd CTL expansion, BALB/c spleen cells were restimulated with SVBALB (Kd Dd) fibroblasts infected with dl754 or dl801 and assayed against SVHTI (Kb Dd) or SVHTG (Kd Db) targets treated with 50 U of gamma interferon (IFN-g) (Genzyme Corp., Framingham, Mass.) for 24 h to overcome the inhibitory effect of Ad E3 gp19K. When viruses that lacked E1A were used, targets were infected with 1,000 PFU per cell 2 days prior to assaying, while all other targets were infected with 100 PFU per cell the day before assaying. All targets were labelled

RESULTS Multiple inoculations lead to the recognition of immunorecessive epitope(s). To confirm that E1A is the immunodominant antigen in the C57BL10/SnJ (B10) strain, B10 mice were inoculated i.p. with Ad5 and assayed against targets that lacked only the E1A region (Fig. 2A). These Ad-specific CTL recognized the E1A transfectant (L13–14) and were unable to lyse the dl312 (DE1A)-infected target which lacked E1A. These results corroborate the findings of Rawle et al. (27) that the immunodominant epitope in B10 mice is E1A and that this response is Db restricted. Since gene therapy applications will likely require multiple administrations of Ad vectors, B10 mice were immunized twice i.p. with wild-type Ad2, and the specificity of the Ad immune response was analyzed. In contrast to the single immunization, CTL were now capable of lysing not only the E1A transfectant (L13–14) but also targets that did not express either E1A or E1B, albeit to a lesser extent (i.e., Ad2/CFTR-2) (Fig. 2B). These data indicate that upon multiple immunizations, not only is the immunodominant CTL population expanded, but a subpopulation of CTL that recognizes antigen(s) that lies outside E1 (E1A and E1B) is activated. A possible solution for circumventing CTL clearance of gene therapy vectors and prolonging transgene expression is the elimination of the immunodominant antigen from the vector. To determine whether eliminating the immunodominant antigen (E1A) would affect the generation of Ad-specific CTL, B10 mice were inoculated once or twice i.p. with either dl312 (DE1A) or Ad2/CFTR-2 (DE1A and E1B). Following a single inoculation with dl312, no Ad-specific CTL were generated (data not shown). In the absence of the immunodominant antigen, multiple immunizations generated an Ad-specific CTL response that was directed against an epitope in a region outside E1 (Fig. 2C). In summary, the CTL response in B10 mice (H-2b) after a single immunization with wild-type Ad was focused on a single immunodominant antigen, E1A. In contrast, multiple immunizations with viruses with or without the immunodominant antigen generated a CTL response against immunorecessive or subdominant antigens. Multiple-i.n. inoculations generate the same Ad-specific immune response as i.p. inoculations. Since the treatment of cystic fibrosis will likely require multiple administrations to the lung, the CTL response was evaluated after double i.n. immu-

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E1A; however, the immunodominant antigen was not defined (27). To map the immunodominant antigen, C3H mice were inoculated once i.p. with Ad5 and assayed against targets that lack the E1 region. CTL generated from Ad immunizations were unable to lyse the Ad2/CFTR-2 (DE1A and E1B) or Ad2/CFTR-7 (DE1A and E1B)-infected targets (Fig. 4A). Since Kk-restricted effectors do not recognize E1A, our results suggest that the Ad-specific CTL are directed against epitope(s) in E1B. To verify this hypothesis, Kk-restricted CTL were assayed against targets lacking either E1A (dl312) or E1B (dl313) only (Fig. 4B). These results demonstrated that Kkrestricted CTL are capable of lysing targets that contained the E1B region (dl309 and dl312), but not targets deleted in E1B (Ad2/CFTR-2, Ad2/CFTR-7, and dl313), confirming that E1B contains the immunodominant Kk-restricted epitope. Immunorecessive epitopes are not recognized after immunizations with vectors that lack the immunodominant Kk epitope. With the knowledge that the immunodominant Kk epitope lies within the E1B region, the question of multiple immunizations and the redirection of the CTL response toward immunorecessive epitopes was investigated. To examine whether CTL generated from multiple immunizations recognize immunorecessive epitopes as had occurred in B10 mice, C3H mice were inoculated twice i.p. or i.n. with wild-type Ad2 or Ad2/CFTR-2 (DE1A and E1B). CTL from both i.p. (Fig. 5A and B) and i.n. (Fig. 6A and B) inoculations failed to recognize immunorecessive epitopes. Even after multiple immunizations, effectors against wild-type Ad responded only to the E1B an-

FIG. 2. The Ad CTL response in C57BL10/SnJ mice following i.p. injection. (A) The CTL response after one injection of Ad5 i.p.; (B) CTL response after two injections of Ad2 i.p.; (C) CTL response after two i.p. injections of Ad2/ CFTR-2. E:T ratio, effector-to-target-cell ratio.

nizations. B10 mice were inoculated twice i.n. with wild-type Ad2 and assayed against targets that express the immunodominant E1A antigen (dl801) or lack E1A/E1B expression (Ad2/ CFTR-2). As was observed following multiple i.p. inoculations with wild-type Ad, the CTL response was directed against E1A (L13–14) and toward an epitope outside E1 (Ad2/CFTR-2) (Fig. 3A). When B10 mice were immunized twice i.n. with Ad2/CFTR-2, which lacks E1, only the non-E1 epitope was recognized (i.e., the E1A transfectant L13–14 was not lysed) (Fig. 3B). The lysis of targets infected with Ad2/CFTR-2 (DE1A and E1B) indicated that at least one of the immunorecessive epitopes was outside of the E1 region. These data indicate that in B10 mice, multiple immunizations either i.p. or i.n. with wild-type or recombinant Ad leads to the generation of CTL that recognize immunorecessive epitopes even in the presence of the immunodominant epitope. Kk-restricted CTL from C3HeB/FeJ mice recognize E1B as the immunodominant antigen. We have shown previously that CTL from C3H mice are Kk-restricted and do not recognize

FIG. 3. The Ad CTL response in C57BL10/SnJ mice after i.n. inoculations. (A) CTL from mice immunized twice i.n. with Ad2; (B) CTL response from mice immunized twice i.n. with Ad2/CFTR-2. E:T ratio, effector-to-target-cell ratio.

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was needed to overcome the Ad E3 gp19K suppression of CTL recognition in Kd-expressing targets. The Ad E3 gp19K protein is capable of binding and retaining the MHC class I molecules of particular haplotypes in the endoplasmic reticulum (1), preventing CTL lysis (29). Gp19K binds strongly to Kd and Db molecules while Dd and Kk are unaffected by gp19K (7). Gp19K did not present an obstacle in C3H targets because gp19K does not bind to the Kk allele. In the B10 (Db) experiments described above, the gp19K effect was overcome by using a cell line in which gp19K does not function to suppress CTL recognition (31). Since gp19K binds strongly to Kd and we have not found a Kd cell line in which gp19K does not function, an alternative method was needed to overcome the gp19K suppression of CTL recognition. A 24-h treatment of targets with IFN-g, which upregulates MHC class I expression, was previously shown to overcome the strong gp19K block in Db cells (31). To verify that this treatment would overcome the gp19K block in Kd-expressing cells, targets were treated with IFN-g for 24 h and assayed against Kd- and Dd-restricted CTL. Treatment of dl309 (gp19K1)infected SVHTG (Kd) targets with IFN-g increased cytolysis by '40-fold, while not altering the lysis of dl327 (DE3)-infected targets (Fig. 7A). Since gp19K does not bind to Dd molecules, IFN-g treatment of SVHTI (Dd) targets increased the lysis of both dl309- and dl327-infected targets equally (Fig. 7B), presumably due, at least in part, to an increase in cell surface

FIG. 4. Large deletion mapping of the antigen recognized by CTL from C3H mice inoculated once i.p. with Ad5. (A) CTL from C3H (H-2Kk) mice assayed against Ad2 E1A/E1B deletion mutants; (B) mapping of the Kk-restricted CTL antigen with Ad5 E1A or E1B deletion mutants. E:T ratio, effector-to-target-cell ratio.

tigen and not to any subdominant epitope(s) in Ad2/CFTR-2 or Ad2/CFTR-7 targets (Fig. 5A and 6A). When Ad2/CFTR-2 was the inoculating agent, there was little or no Ad-specific CTL response, indicating that no immunorecessive epitopes were recognized (Fig. 5B and 6B). Considering that the in vitro stimulating cell could be altering the subdominant response, wild-type and Ad2/CFTR-2-primed splenocytes were restimulated in vitro with PSC3H fibroblasts infected with Ad2/ CFTR-2 (DE1A and E1B) instead of dl801. In neither case were CTL against subdominant epitopes detected (data not shown). Only after inoculation of Ad2/CFTR-2 three times i.n. was a minimal but sporadic Ad-specific response detected (data not shown). Therefore, in contrast to the phenomenon observed with B10 mice (H-2b), C3H mice (H-2k) had diminished ability to recognize subdominant epitopes even after multiple immunizations. Treatment of BALB/c target cells with IFN-g is necessary to overcome the E3-gp19K block to CTL recognition. To investigate whether the generation of immunorecessive epitopes is generalizable to other mouse strains, multiple immunization protocols were applied to BALB/c mice. Before examination of the subdominant response, the immunodominant epitope was delineated. We have previously reported that the immune response to Ad in BALB/c mice is both Kd and Dd restricted and is not directed against E1A (27). To map the epitope(s) recognized in BALB/c mice, a modification of the CTL protocol

FIG. 5. The generation of Ad-specific, Kk-restricted CTL after i.p. immunization. (A) C3H mice immunized twice i.p. with Ad2; (B) C3H mice immunized twice i.p. with Ad2/CFTR-2. E:T ratio, effector-to-target-cell ratio.

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Although there are similarities between C3H Kk- and BALB/c Dd-restricted responses, the lack of a Dd immunorecessive response is confounded by a strong Kd response which could mask a Dd-subdominant response (12). The Kd-restricted CTL response after i.p. inoculations was mapped in a manner similar to that for the Dd response, except that the targets were SVHTG (Kd Db) fibroblasts. Targets infected with viruses that lack the E1 region (Ad2/CFTR-2) were lysed by Kd-restricted CTL (Fig. 10). Thus, the antigen recognized by Kd-restricted CTL is outside the E1 region. Kd-restricted effectors also lysed mutants lacking E3 or E4 regions (data not shown), suggesting that this response is directed toward epitopes within E2 or the late region or that there are multiple Kd epitopes in several different regions. Currently, no deletion mutants are available to define further the immunodominant Kd antigen. DISCUSSION This report demonstrates that CTL from mice immunized with wild-type Ad recognize an immunodominant epitope(s) within E1A in association with Db, epitopes within E1B in association with Kk and Dd, and non-E1 antigen(s) in associa-

FIG. 6. The generation of Ad-specific, Kk-restricted CTL after i.n. immunization. (A) C3H mice immunized twice i.n. with Ad2; (B) C3H mice immunized twice i.n. with Ad2/CFTR-2. E:T ratio, effector-to-target-cell ratio.

expression of the Dd molecule. Overcoming gp19K’s suppression of CTL recognition was necessary because the many of the deletion mutants used in this study are on a gp19K1 background. Ad-specific CTL in BALB/c mice recognize E1B as the immunodominant Dd epitope, and an antigen outside E1 is immunodominant for Kd. To map the epitope recognized by Ddrestricted CTL, BALB/c mice were immunized i.p. with Ad5 and restimulated in vitro with SVBALB (H-2d) fibroblasts infected with dl754 (Dgp19K). The Dd-restricted CTL response was analyzed by assaying the CTL against SVHTI (Kb Dd) targets infected with E1A/E1B deletion mutants. The Dd-restricted CTL recognized dl312 (DE1A) targets, while elimination of the entire E1 region (Ad2/CFTR-2 and Ad2/CFTR-7) abolished CTL lysis (Fig. 8). These data indicate that E1B contains the immunodominant antigen for the Dd allele. Knowledge of the location of the immunodominant antigen for the Dd-restricted response allowed us to address whether immunorecessive epitopes are recognized upon multiple inoculations. To address this, BALB/c mice were immunized twice i.n. with wild-type Ad2 or Ad2/CFTR-2 and assayed against various targets. As with C3H mice, multiple immunizations did not induce the recognition of immunorecessive epitopes outside the E1A/E1B region (Fig. 9 A and B). Dd-restricted CTL from multiple Ad2 immunizations recognized cells expressing E1B (Fig. 9A) and when Ad2/CFTR-2 was used as the immunizing agent, no Ad-specific CTL were generated (Fig. 9B).

FIG. 7. IFN-g treatment of targets overcomes gp19K suppression of CTL lysis. BALB/c effectors were generated by i.p. injection of Ad5 once and restimulation in vitro with SVBALB (Kd Dd) fibroblasts infected with dl754 (gp19K2). Target cells were either untreated or treated with 50 U of IFN-g or were mock treated 24 h prior to assay. (A) SVHTG (Kd Db) targets; (B) SVHTI (Kb Dd) targets. E:T ratio, effector-to-target-cell ratio.

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FIG. 8. Mapping of the Dd CTL epitope. BALB/c mice were immunized i.p. with Ad5, and spleen cells were restimulated in vitro. SVHTI (Kb Dd) targets were infected with large E1A/E1B deletion mutants. E:T ratio, effector-totarget-cell ratio.

d

tion with K . Upon multiple immunizations with wild-type virus or viruses lacking the immunodominant epitope, subdominant epitopes are recognized in B10 (Db), but not in C3H or BALB/c (Dd allele) mice (Table 1). The location of the Dbrestricted subdominant epitopes within the Ad genome cannot be defined by available mutants, but it does not reside within

FIG. 10. Mapping of the epitope recognized by Kd-restricted CTL. BALB/c mice were inoculated i.p. with Ad5, and the in vitro-restimulated effectors were assayed against virally infected SVHTG (Kd Db) targets. E:T ratio, effector-totarget-cell ratio.

E1, E3, or E4 (data not shown). For the Kk allele in C3H mice and the Dd allele in BALB/c mice, eliminating the immunodominant antigen (E1B) from the inoculating virus effectively eliminates the CTL response. These data suggest that the strategy of modifying gene therapy vectors to remove CTL epitopes would be effective in some individuals but not in others after multiple inoculations. In the experiments reported here, regions of the virus containing CTL epitopes were identified by deletion mapping. That is, target cells were infected with viruses that delete regions of the viral genome and tested for loss of CTL recognition. Several assumptions underlie this approach. First, it was assumed that the CTL response would be directed against one or a very few dominant epitopes. Such preferential responses to dominant epitopes despite the apparent availability of many potential epitopes have been seen with other virus systems (e.g., see references 22, 25, and 26), although the mechanism of epitope dominance remains unknown. Were CTL to recognize many different epitopes from the viral genome, cells infected with deletion mutants lacking only some would not be distinguished from the wild-type in sensitivity to CTL lysis. In earlier studies, we found that after a single immunization with Ad, B10 mice produced CTL that recognized only epitopes within E1A (27), suggesting that CTL to Ad would be relatively restricted in their epitope selection.

TABLE 1. Summary of mouse CTL responses to group C Ads Dominant epitope(s)

Mouse type

FIG. 9. Immunorecessive epitopes are not detected in BALB/c mice. BALB/c mice were immunized twice i.n., and the in vitro restimulated effectors were assayed against virally infected SVHTI (Kb Dd) targets. (A) In vivo immunization with Ad2; (B) in vivo immunization with Ad2/CFTR-7. E:T ratio, effector-to-target-cell ratio.

C57BL/10 (H-2b) Kb Db C3H (H-2k) Kk Dk BALB/c (H-2d) Kd Dd a

—, undetermined.

Immunorecessive epitope(s)

None E1A

None Non-E1, -E3, and -E4

E1B None

6None None

Non-E1, -E3, and -E4 E1B

—a None

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Second, it was assumed in cases in which CTL failed to lyse mutant-infected cells that the region deleted from the virus contained the epitope(s) recognized by CTL. During virus infection, transcription of many viral genes depends on prior expression of other viral proteins, notably those encoded within E1A (30). Expression of E1A proteins is required for the timely transcription of all other viral genes after infection (24). Nonetheless, there are several reasons to believe that deleting E1A does not lead to functional removal of CTL epitopes derived from genes outside E1A. First is our previous observation (27), also reported here, that cells infected with dl312, which lack all E1A transactivation functions, are recognized by CTL from C3H and BALB/c mice. That these CTL are not recognizing residual E1A epitopes was confirmed by their failure to lyse transfected syngeneic cells expressing E1A (27). Therefore, some non-E1A genes must be expressed at levels sufficient to permit CTL recognition. In our studies, infections with E1A-deleted viruses were performed at high multiplicity of infection and allowed proceeding for 40 to 48 h, under which conditions viral mRNAs (24) and early proteins (9) slowly accumulate. E1-deleted viruses have even been shown to transcribe late genes in infected mouse lungs (41). Surprisingly, we find that CTL recognize epitopes from E1Adeleted viruses at even very early times postinfection (not shown). The reason for this apparent paradox lies in the exquisite sensitivity of CTL to viral peptides. Kageyama et al. (19) have reported that some CTL require expression of fewer than 10 peptide-MHC molecules per target cell for recognition and cytolysis to occur. Thus, even very low levels of virus gene expression are sufficient to produce a CTL target. Indeed, CTL have been shown to recognize epitopes derived from genes with no promoter or translation start site or after a termination codon (5, 21). Thus, the levels of Ad proteins produced in infected cells, even under conditions in which their normal regulation has been interrupted due to the lack of regulatory functions such as E1A, will certainly be sufficient to allow CTL recognition. Our findings on E1B immunodominance for the Kk allele are in apparent contradiction to reports by Zhang et al. (45) and Yang et al. (41). Zhang et al. mapped the immunodominant epitope in CBA (H-2k) mice to E1A, but we (reference 27 and this report) find that Kk-restricted CTL do not recognize an E1A transfectant and do lyse targets infected with dl312 which lacks E1A. In addition, Yang et al. generated Ad-specific CTL in CBA mice (H-2k) after immunizing only once i.n. with a CFTR vector that lacked E1. In our study, C3H (H-2k) mice did not mount a CTL response against Ad CFTR vectors even after two i.n. inoculations. One possible explanation for these apparent discrepancies is that genetic differences between CBA and C3H mice or differences in the vector backbone (20) resulted in different CTL responses. As we have seen with the CTL response in Ad-infected B10 and B6 mice (36a), even substrains of mice may have slightly different immunodominant responses. Another possible explanation is that differences between in vitro stimulators could lead to the expansion of different CTL populations. Our secondary in vitro stimulators were Ad-infected, SV-40 transformed fibroblasts, while both of the other groups used Ad-infected splenocytes as stimulators. This could have led to the generation of CD41 class I-restricted killers (42) or to the expansion of CTL that recognize E1A instead of E1B. Whether subtle differences in antigen processing and presentation could explain these differences remains to be tested. The findings from this study offer some hope for modification of Ad gene therapy vectors. It may be possible to alter the CTL response (as in C3H mice or Dd in BALB/c mice) by

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creating vectors that lack the immunodominant antigen or vectors with altered anchor-subanchor residues in the antigenic regions. Of course, the CTL response is likely to vary within the diverse human population, thus making epitope mapping more complicated. Even in the absence of the immunodominant antigen(s), repeat administrations could cause the generation of CTL against subdominant epitopes, as was observed with B10 mice. Another possible method for preventing CTL recognition would be alterations in the Ad genome such that only minimal levels of Ad proteins are produced (i.e., DE2 [10, 11] or DE4 [2]). Although very few peptide-MHC complexes are necessary for CTL lysis, the requirement for CTL activation and expansion depends on the accessory molecules and cell type of the presenting cell (32). This could vary depending on the route (intravenous verse i.n.) and dose. We have shown that a single i.p. inoculation in B10 mice leads to the expansion of CTL that recognize E1A as the immunodominant antigen, while others (20, 38) have shown that a larger i.n. dose leads to the generation of CTL that also recognize the subdominant non-E1 antigen. Only after multiple i.p. inoculations were CTL that recognized the subdominant epitope generated. Thus, it seems that lower doses mulitple times or a single larger dose can lead to the recognition of subdominant antigens. Expression vectors that minimize the expression of viral antigens could prevent the threshold needed for CTL activation and expansion. These approaches are currently being investigated. ACKNOWLEDGMENTS We thank William S. M. Wold, Terry W. Hermiston, and David Ornellas for providing the viruses used in this study. We also thank Jennifer Hull for excellent technical assistance. This work was funded by NIH grant CA58736 and a grant from Genzyme Corp. REFERENCES 1. Anderson, M., S. Paabo, T. Nilsson, and P. Peterson. 1985. Impaired intracellular transport of class I MHC antigens as a possible means for adenovirus to evade immune surveillance. Cell 43:215–222. 2. Armentano, D., C. C. Sookdeo, K. M. Hehir, R. J. Gregory, J. A. St. George, G. A. Prince, S. C. Wadsworth, and A. E. Smith. 1995. Characterization of an adenovirus gene transfer vector containing an E4 deletion. Hum. Gen. Ther. 6:1343–1353. 3. Bennink, J. R., J. W. Yewdell, G. L. Smith, and B. Moss. 1986. Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes. J Virol. 57:786–791. 4. Challberg, S. S., and G. Ketner. 1981. Deletion mutants of adenovirus 2: isolation and initial characterization of virus carrying mutations near the right end of the viral genome. Virology 114:196–209. 5. Chomez, P., E. De Plaen, A. Van Pel, C. De Smet, J.-P. Szikora, C. Lurquin, A.-M. Lebacz-Verheyden, and T. Boon. 1992. Efficient expression of tum2 antigen P91A by transfected subgenic fragments. Immunogenetics 35:241– 252. 6. Clayman, G. L., A. K. el-Naggar, J. A. Roth, W. W. Zhang, H. Goepfert, D. L. Taylor, and T. J. Liu. 1995. In vivo molecular therapy with p53 adenovirus for microscopic residual head and neck squamous carcinoma. Cancer Res. 55:1–6. 7. Cox, J. H., J. W. Yewdell, L. C. Eisenlohr, P. R. Johnson, and J. R. Bennink. 1990. Antigen presentation requires transport of MHC class I molecules from the endoplasmic reticulum. Science 247:715–8. 8. Doherty, P., W. Allan, and M. Eichelberger. 1992. Roles of ab and gd T cell subsets in viral immunity. Annu. Rev. Immunol. 10:123–151. 9. Duerksen-Hughes, P., W. S. Wold, and L. R. Gooding. 1989. Adenovirus E1A renders infected cells sensitive to cytolysis by tumor necrosis factor. J. Immunol. 143:4193–4200. 10. Engelhardt, J. F., L. Litzky, and J. M. Wilson. 1994. Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a. Hum. Gene Ther. 5:1217–1229. 11. Engelhardt, J. F., X. Ye, B. Doranz, and J. M. Wilson. 1994. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. USA 91:6196– 6200. 12. Gooding, L. R. 1980. Anomalous behavior of H-2Kb in immunity to syngeneic SV40 transformed cells: evidence for cytotoxic T cell recognition of

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