HLA-A*0201, HLA-A*1101, and HLA-B*0702 Transgenic Mice Recognize Numerous Poxvirus Determinants from a Wide Variety of Viral Gene Products

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1 This information is current as of September 3, References Subscription Permissions Alerts Errata HLA-A*0201, HLA-A*1101, and HLA-B*0702 Transgenic Mice Recognize Numerous Poxvirus Determinants from a Wide Variety of Viral Gene Products Valerie Pasquetto, Huynh-Hoa Bui, Rielle Giannino, Fareed Mirza, John Sidney, Carla Oseroff, David C. Tscharke, Kari Irvine, Jack R. Bennink, Bjoern Peters, Scott Southwood, Vincenzo Cerundolo, Howard Grey, Jonathan W. Yewdell and Alessandro Sette J Immunol 2005; 175: ; ; doi: /jimmunol This article cites 45 articles, 28 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: An erratum has been published regarding this article. Please see next page or: /content/175/12/ full.pdf Downloaded from by guest on September 3, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2005 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 HLA-A*0201, HLA-A*1101, and HLA-B*0702 Transgenic Mice Recognize Numerous Poxvirus Determinants from a Wide Variety of Viral Gene Products 1 Valerie Pasquetto,* Huynh-Hoa Bui,* Rielle Giannino,* Fareed Mirza, John Sidney,* Carla Oseroff,* David C. Tscharke, Kari Irvine, Jack R. Bennink, Bjoern Peters,* Scott Southwood, Vincenzo Cerundolo, Howard Grey,* Jonathan W. Yewdell, and Alessandro Sette 2 * In virus models explored in detail in mice, CTL typically focus on a few immunodominant determinants. In this study we use a multipronged approach to understand the diversity of CTL responses to vaccinia virus, a prototypic poxvirus with a genome 20-fold larger than that of the model RNA viruses typically studied in mice. Based on predictive computational algorithms for peptide binding to HLA supertypes, we synthesized a panel of 2889 peptides to begin to create an immunomic map of human CTL responses to poxviruses. Using this panel in conjunction with CTLs from vaccinia virus-infected HLA transgenic mice, we identified 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA-B*0702-restricted CD8 T cell determinants distributed over 20 distinct proteins. These peptides were capable of binding one or multiple A2, A3, and B7 supertype molecules with affinities typical of viral determinants. Surprisingly, many of the viral proteins recognized are predicted to be late gene products, in addition to the early intermediate gene products expected. Nearly all of the determinants identified have identical counterparts encoded by modified vaccinia virus Ankara as well as variola virus, the agent of smallpox. These findings have implications for the design of new smallpox vaccines and the understanding of immune responses to large DNA viruses in general. The Journal of Immunology, 2005, 175: *La Jolla Institute for Allergy and Immunology, San Diego, CA 92109; Tumor Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford, United Kingdom; Epimmune Incorporated, San Diego, CA 92121; Laboratory of Viral Diseases, National Institutes of Health, Bethesda, MD 20892; and Division of Immunology and Infectious Diseases, Queensland Institute of Medical Research, Herston, Queensland, Australia Received for publication June 6, Accepted for publication July 27, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Contract HHSN C and Grant RO1AI56268 from the National Institutes of Health. V.C. and F.M. are recipients of grants from the Cancer Research United Kingdom (C399-A2291) and United Kingdom Medical Research Council. D.C.T. is the recipient of the National Health and Medical Research Council of Australia Howard Florey Centenary Fellowship (224273). 2 Address correspondence and reprint requests to Dr. Alessandro Sette, La Jolla Institute for Allergy and Immunology, 3030 Bunker Hill Street, Suite 326, San Diego, CA address: alex@liai.org 3 Abbreviations used in this paper: HBV, hepatitis B virus; HCV, hepatitis C virus; ORF, open reading frame; VARV, variola virus; WR, Western Reserve; VACV, vaccinia virus WR strain; SI, stimulation index; SFC, spot-forming cell; MVA, vaccinia virus Ankara. Immunodominance is defined as the phenomenon whereby only a small fraction of all of the possible determinants from a particular pathogen elicit an immune response in an infected individual (1, 2). Immunodominance can be profound, resulting in CTL induced by only one to a few determinants dominating the response to a pathogen encoding thousands of potential determinants. Such extreme immunodominance is frequently observed in immune responses to various mouse viruses (3). However, it has been shown that human responses to infections with different viruses (Flu, HIV, CMV, hepatitis B virus (HBV), 3 hepatitis C virus (HCV)) tend to be multispecific (directed against multiple proteins derived from a given pathogen) and broad (directed against multiple determinants within a given protein) (4, 5). Furthermore, nearly all viruses extensively studied in humans and mice contain relatively small genomes (10 20 kb). It is impossible to predict the complexity of human responses to viruses with more coding capacity, such as poxviruses, with a genome of 200 kb encoding 200 identifiable nonoverlapping open reading frames (ORFs). Recent concerns that variola virus (VARV), the agent of smallpox, could potentially be reintroduced into nature as a bioterrorism weapon, as well as recent outbreaks of zoonotic Orthopoxvirus infections, have led to renewed interest in poxvirus vaccination (6). Eradication of smallpox was based on immunization with vaccinia virus Western reserve (WR) strain (VACV), a poxvirus of unknown origin, with an uncertain relationship to Jenner s vaccine, and whose identity remains shrouded in mystery. Although smallpox eradication is perhaps the greatest public health accomplishment in human history (6), VACV has a number of serious shortcomings as a modern day vaccine (7). It is therefore of great importance to develop new improved vaccines that protect against smallpox, but with less adverse reactions and diminished potential for transmission from vaccinees to others. The VACV and VARV genomes are 90% homologous. It is believed that both T and B cell responses directed to identical or highly homologous target sequences play a vital role in heterologous protection against VARV afforded by vaccination with vaccinia virus. Several lines of evidence suggest that cellular immunity makes an important contribution to smallpox immunity (8 12). Ultimately, it will be impossible to ascertain the precise mechanisms that contribute to protective VARV immunity, and improved vaccines will have to be based on the use of modified forms of live VACV immunization. The efficacy of such vaccines will have to be based on their abilities to recapitulate responses Copyright 2005 by The American Association of Immunologists, Inc /05/$02.00

3 5505 induced by Dryvax and other VACV preparations that were shown to offer good immunity to smallpox. Thus, in addition to its intrinsic interest in terms of understanding immunodominance in humans to large viruses, it is critical to characterize the HLA-restricted T cell responses to VACV. To date, reports have been limited to the description of just a few determinants presented by a single restricting MHC class I molecule (13 15). Specifically, Ennis and coworkers (14) reported the identification of two different HLA-A*0201-restricted epitopes induced by vaccinia vaccination and conserved among vaccinia and VARVs. Drexler and coworkers (13) reported the identification of an HLA-A*0201-restricted epitope using a combination of bioinformatics predictions and assay in HLA transgenic mice. The paucity of information stems partly from the daunting size of the poxvirus genome and difficulties in obtaining samples from vaccinees. To deal with the large poxvirus coding capacity, we have used computer algorithms to predict potential immunogenic peptides. Previous work from numerous systems has established that the greatest single hurdle to immunogenicity for potential viral determinants is their affinity for class I molecules (3). Generally, only 1% of peptides are able to bind any given MHC class I allomorph above the threshold affinity associated with immunogenicity (K D 500 nm). In the present study, we tackle this problem by using a computational approach to predict HLA-binding peptides encoded in the VACV genome. Although predictive algorithms are not perfect, they successfully identify 80% of class I binding peptides. To this end, we generated a panel of 2889 synthetic peptides. To surmount problems with obtaining VACV-specific CTLs from human volunteers, we used HLA transgenic mice. Numerous studies have shown that antiviral CTL responses in humans and HLA transgenic mice overlap to a remarkable degree (16 20). This is consistent with the substantial literature documenting that the Ag-processing machinery is highly conserved between mouse and human cells (21, 22). In this study we describe how this approach has resulted in the identification of 20 new antigenically active determinants that bind to HLA molecules with high affinity. Materials and Methods Peptides Peptides used in initial screening experiments were tested without chromatographic purification and were synthesized as crude material by Mimotopes, Pepscan Systems BV, or A and A Labs, as described elsewhere (23). Candidate epitopes identified in pool deconvolution studies, or peptides use as radiolabeled ligands for binding assays, were resynthesized by A and A Labs and purified to 95% homogeneity by reverse-phase HPLC. Purity of these peptides was determined using analytical reverse-phase HPLC and amino acid analysis, sequencing, and/or mass spectrometry. Peptides were radiolabeled with the chloramine T method, as described elsewhere (24). MHC peptide-binding assays Quantitative assays to measure the binding affinity of peptides to purified HLA-A2- (A*0201, A*0202, A*0203, A*0206, A*6802), -A3- (A*0301, A*1101, A*3101, A*3301, A*6801), and -B7- (B*0702, B*3501, B*5101, B*5301, B*5401) supertype molecules are based on the inhibition of binding of a radiolabeled standard peptide, and were performed as previously described (23 26). Briefly, 1 10 nm of radiolabeled peptide was coincubated at room temperature with 1 M to 1 nm purified MHC in the presence of 1 3 M human 2 -microglobulin (Scripps Laboratories) and a mixture of protease inhibitors. After a 2-day incubation, binding of the radiolabeled peptide to the corresponding MHC class I molecule was determined by capturing MHC/peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-One) coated with the W6/32 Ab, and measuring bound cpm using the TopCount microscintillation counter (Packard Instrument). For competition assays, the concentration of peptide yielding IC 50 of the binding of the radiolabeled peptide was calculated. Peptides were typically tested at six different concentrations covering a 100,000-fold dose range, and in three or more independent assays. Under the conditions used, where (label) (MHC) and IC 50 (MHC), the measured IC 50 values are reasonable approximations of the true K d values. Bioinformatic analyses An unpublished VACV (WR) sequence provided by Dr. Bernard Moss (National Institute of Allergy and Infectious Diseases), which has been the basis of other studies (27, 28), has been used for this study. To identify candidate epitopes for use in ORF-specific peptide pools, each predicted ORF of the vaccinia WR strain was analyzed using previously described algorithms (4) that predict the affinity of peptides for specific HLA class I molecules. The panel of algorithms used allowed prediction of ligands for HLA-A2, -A3, and -B7-supertype molecules. For each gene, peptides predicted to bind with an IC nm were selected for study. To reduce the number of predicted peptides identified in large ORFs, predictions for all supertypes were combined, and the best scoring 40 candidates, regardless of supertype, were selected. As a result, 964 A2-supertype, 1584 A3- supertype, and 341 B7-supertype peptides were selected and synthesized for study. Mice HLA-A*0201/K b, HLA-A*1101/K b, and HLA-B*0702/K b transgenic mice used in this study were the F 1 generation derived from crossing homozygous transgenic mice (H-2 b haplotype) expressing a chimeric gene consisting of the 1 and 2 domains of HLA and the 3 domain of H-2K b (17 19) with BALB/c mice (The Jackson Laboratory). HHD A 2 mice (29) were bred at the Weatherall Institute of Molecular Medicine. The other transgenic mice were bred and maintained at the La Jolla Institute for Allergy and Immunology facility (San Diego, CA) following National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved animal protocols. Stimulator cells and cell lines in ELISPOT assays Stimulator cells used for peptide-specific IFN- release were Jurkat cells transfected with the HLA-A*0201/K b or the HLA-B*0702/K b chimeric genes, or the.221a*1101/k b cell line (17, 18, 30). These cell lines were transfected with the same HLA construct expressed in the corresponding HLA transgenic mice used in this study. The cell line.221a*1101/k b was derived by transfection of the HLA-A*1101/K b gene into 3A tumor cells, which lacks expression of HLA-A, -B, or -C class I genes due to gamma-ray-induced deletions in the HLA complex (31). LPS-stimulated B lymphoblasts obtained by cultivating splenocytes in the presence of LPS (8.5 g/ml) and dextran sulfate (7 g/ml) (Sigma-Aldrich), for 3 days at 37 C, were also used as stimulator cells. All cells were grown in RPMI complete culture medium (RPMI 1640 medium, 25 nm HEPES (ph 7.4; Invitrogen Life Technologies), supplemented with 10% FBS, 4 mm L- glutamine, M 2-ME, 0.5 mm sodium pyruvate, 0.1 mm MEM nonessential amino acids, 100 g/ml streptomycin, and 100 IU/ml penicillin). Viruses The WR strain of vaccinia virus was obtained from Dr. Bernard Moss (National Institute of Allergy and Infectious Diseases). Infection and immunizations HLA transgenic mice were infected i.p. with PFU of vaccinia WR strain (VACV) in PBS. After 7 days, the mice were sacrificed, and the splenocytes or purified CD8 T cells were used for ex vivo mouse-ifn- enzyme-linked immunospot (ELISPOT) measurement. For peptide immunization, mice were immunized s.c. with a mixture of peptide (10 g/ mouse) and the helper IA-b-restricted epitope, HBV core (140 g/mouse) (32) in PBS/10% DMSO emulsified in IFA. After days, the mice were sacrificed, and CD8 -purified T cells were used for ex vivo ELISPOT measurement. Ex vivo ELISPOT assays The ELISPOT assays were performed as previously described (33). Briefly, either splenocytes or splenic CD8 T cells (isolated by anti-cd8-coated magnetic beads (Miltenyi Biotec)) were cultured with peptide pulsed or VACV WR-infected stimulator cells. The stimulator cells were Jurkat-A*0201/K b, Jurkat-B*0702/K b cells, A*1101/K b,or LPS blasts, in flat-bottom 96-well nitrocellulose plates (Immobilon-P membrane; Millipore), which had been precoated with anti-ifn- mab (BD Pharmingen; 4 g/ml). After 20-h

4 5506 POTENTIAL HLA-RESTRICTED VACCINIA VIRUS-DERIVED EPITOPES incubation at 37 C, plates were washed with PBS/0.05% Tween, and wells were incubated with biotinylated anti-ifn- mab (BD Pharmingen; 1 g/ ml) for 4 h at 37 C. After additional washing, spots were developed by sequential incubation with Vectastain ABC peroxidase (Vector Laboratories) and 3-amino-9-ethylcarbazole solution (Sigma-Aldrich) and counted by computer-assisted image analysis (Zeiss KS ELISPOT Reader). Each assay was performed in six replicate wells, and the experimental values were expressed as the mean net spots/10 6 unfractionated splenocytes or CD8 lymphocytes SEM for each peptide. Responses against irrelevant peptides (HCV core 132, DLMGYIPLV for A*0201; HBV env. 378, LLPIFFCLWV for B*0702; and Human Mage3 69, SSLPTTMNY for A*1101) were measured to establish background values that were subtracted from the experimental values. To determine the level of significance, a Student s t test was performed in which p 0.05 using the mean of triplicate values of the response against relevant peptides vs the response against irrelevant control peptides. The net number of spots/10 6 effector cells was calculated as follows: [(number of spots against relevant peptide) (number of spots against irrelevant control peptide)] [( )/ (number of effector cells/well)]. The stimulation index (SI) was calculated as follows: (number of spots against relevant peptide)/(number of spots against irrelevant control peptide). Results Prediction of HLA-determinant candidates from the VACV genome In previous studies, we demonstrated that 95% of HLA-restricted determinants bind their relevant HLA molecules with a K D of 500 nm or less (34). Subsequent studies described computer algorithms that allow the prediction of the binding affinity of potential determinants to specific HLA molecules (4). In using these algorithms, different affinity thresholds can be selected. High-affinity thresholds generate highly accurate predictions at the cost of capturing a small fraction of determinants. Lower affinity thresholds capture a progressively larger fraction of the determinants, at the expense of lower accuracy (i.e., more false predictions are generated). In this study, we use predictive thresholds previously established to predict 75% of HLA-restricted determinants (4). A large fraction of HLA class I allomorphs can be assigned into a relatively few supertypes, each characterized by largely overlapping peptide-binding repertoires reflecting consensus structures in the main peptide binding pockets (35). Predictions were performed for the HLA-A2, -A3, and -B7 supertypes. At least one of these three supertype specificities is present in 85% of the human worldwide population among all ethnic backgrounds (34). These algorithms were applied to the 258 predicted ORFs in the complete VACV-WR genome. Although not all of these ORFs are known to be expressed, we included all of them, because it has been shown that poorly expressed or aberrant transcripts can yield T cell determinants (36). We synthesized 964 HLA-A2, 1584 HLA-A3, and 341 HLA-B7 potential ligands from the poxvirus proteome based on in silico prediction of high or intermediate binding. Identification of 21 different VACV-derived determinants We determined the antigenicity of synthetic peptides using splenocytes obtained from HLA transgenic mice 1 wk after i.p. infection with VACV. The derivation of HLA-A*0201/K b and HLA- B*0702/K b mice has been described elsewhere (17, 19). To identify HLA-A3 supertype peptides, we generated HLA-A*1101/K b transgenic mice (18). Splenocytes were incubated ex vivo with Jurkat HLA-A*0201/K b -, Jurkat HLA-B*0702/K b -, and.221- A*1101/K b -transfected cell lines, respectively, that had been pulsed with pools of the corresponding potentially antigenic peptides (each pool contained 15 peptides), scoring antigenicity by ELISPOT assay for IFN- secretion. The results from individual experiments were highly reproducible, and an average of 2 3 independent experiments were used to identify antigenically active pools. The results of these initial screens are shown in Fig. 1 for HLA- A*0201/K b, -A*1101/K b, and -B*0702/K b transgenic mice, respectively. Peptide pools generating an average of 20 spot-forming cells (SFCs)/10 6 cells (shown in black in Fig. 1) and an average SI 1.4 were selected for further characterization experiments. This low SI threshold was purposely selected to allow for a comprehensive identification, and is on the side of inclusiveness in the epitope identification process. None of the pools elicited positive responses from naive mice. In total, 24 of 81 HLA-A2, 6 of 103 HLA-A3, and 7 of 34 HLA-B7 supertype-restricted pools generated positive responses following VACV infection. Using transfected cell lines, we next identified the individual peptides in each pool that were responsible for the antigenic activity detected. As expected, based on the low criteria for pool positiveness used, as described above, for several of the pools that gave weak responses, no definitive individual peptide could be identified that stimulated a response. However, we also were able to identify 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA- B*0702-restricted determinants that gave reproducibly robust signals (Fig. 2) (SI above 2 in multiple independent experiments). One of these epitopes was previously and independently been identified by Drexler et al. (13). Characterization of HLA-A*0201-restricted determinants Having identified a number of candidate epitopes, we needed to demonstrate that they were truly recognized by T CD8 restricted by the presumed HLA gene product. The 14 determinants identified in VACV WR-infected HLA-A*0201 transgenic mice were tested by using APCs expressing various mouse or human MHC class I molecules and varying doses of the peptide determinants to stimulate purified CD8 T cells from the HLA-A*0201/K b -immunized mice. Specifically, we used LPS-stimulated B lymphoblasts from the HLA-A*0201/K b transgenic CB6F1 mice; LPS B cell blasts from CB6F1 mice, which do not express the HLA transgene, and Jurkat HLA-A*0201/K b, a human cell line transfected with the same HLA-A*0201/K b construct expressed in the HLA transgenic mice. Additional controls included LPS blasts from the unrelated mouse strain CBA/J (H-2 k haplotype), as well as LPS blasts derived from K b D b knockout mice, which do not express any classical MHC class I molecules. All 14 determinants were able to activate highly purified T CD8 (Fig. 3, and Table I). Two patterns of MHC restriction were observed. Eleven peptides demonstrated robust antigenicity using HLA-A*0201/K b Jurkat cells and HLA-A*0201/K b transgenic B blasts, but not with any of the APCs not expressing the HLA- A*0201 transgene (Fig. 3, A and B, show representative examples). For three other determinants (M1L, ; A17L, 61 70; and B6R, ) a more complex pattern was seen. These peptides were antigenic when presented by either HLA-A*0201/K b - expressing target cells or CB6F1 B blasts, but not to peptidepulsed LPS blasts derived from the control H-2 k mice or class I knockout mice (see Fig. 3, C and D, for representative data). Thus, these peptides are presented by either HLA-A*0201 or one of the endogenous mouse class I coexpressed in the HLA-A*0201/K b transgenic mice. All of the peptides tested were able to activate T CD8 responses in range of M (Fig. 3, and data not shown). This is typical of peptides that represent determinants presented to the immune system by processing of viral Ags.

5 5507 FIGURE 1. Screening of pools of HLA-predicted binding peptides derived from VACV sequences. Positive pools of peptides selected for further analysis are indicated in black. The criteria of positivity were SI 1.4 and net SFCs/10 6 cells 20. A, A total of 24 of 81 pools was identified in VACV-infected HLA-A*0201/K b transgenic mice. B, A total of 6 of 103 pools was identified in VACV-infected HLA-A*1101/K b transgenic mice. C, A total of 7 of 34 pools was identified in VACV-infected HLA-B*0702/K b transgenic mice. Immunogenicity of the HLA-A*0201-restricted determinants in various mice strains We extended these studies to purified T CD8 obtained from VACV WR-infected mice of various MHC haplotypes for the ability to respond to the antigenic peptides presented by autologous LPS blasts (Table I). As expected, we failed to detect responding T CD8 to any of the peptides in CBA/J mice, or in H-2K b / D b / mice, which lack expression of any MHC class I molecules. Concordant with the results described above, 11 of the 14 (78.6%) HLA-A*0201-restricted peptides were only recognized by mice expressing HLA-A*0201. For nine of these determinants, vigorous responses were also observed in HHD A 2 mice, which only express HLA-A*0201 (i.e., not K b or D b ). Interestingly, two peptides (G7L ( ) and VACWR050 ( )) were recognized by HLA-A*0201/K b T CD8 but not by HHD (or CB6F1) T CD8. These data suggest that the presence of mouse class I MHC molecules are required to generate the T CD8 repertoire capable of responding to these peptides. The three peptides for which both mouse and human restriction was demonstrated above were recognized by T CD8 from CB6F1 mice, further confirming that these determinants can be restricted by the endogenous mouse class I molecules as well as the human HLA-A*0201 transgene. Responses to two of the peptides (M1L ( ) and A17L (61 70)) were generated in both HHD and CB6F1 mice, clearly demonstrating this dual specificity. The B6R ( ) determinant was immunogenic in CB6F1 and the HLA- A*0201/K b transgenic mice, but not in HHD mice. As postulated above, this suggests that mouse class I expression was required for the generation of a T cell repertoire capable of recognizing this determinant in the context of HLA-A*0201 as well as in the context of mouse

6 5508 POTENTIAL HLA-RESTRICTED VACCINIA VIRUS-DERIVED EPITOPES FIGURE 2. Screening of individual HLA-predicted binding peptides from immunogenic ORFs derived from VACV. Positive peptides selected are indicated in black. The criteria of positivity were SI 2.0 and net SFCs/10 6 cells 20. An asterisk indicates epitopes for which a second overlapping peptide containing the same core sequence also induced a positive response. The amino acid sequence and the protein name from which each of the epitopes was derived are indicated. A, Fourteen positive peptides were identified in HLA-A*0201/K b transgenic mice. B, four positive peptides were identified in HLA-A*1101/K b transgenic mice. C, Three positive peptides were identified in HLA-B*0702/K b transgenic mice. class I. Notably, the sum of T CD8 responses to the 14 individual peptides in the various mouse strains tested was equal to or exceeded the magnitude of the responses detected against VACV-infected target cells. This suggests that a significant proportion of the total response to the virus can be accounted for by the response to these determinants. In the same set of experiments, we tested a previously reported HLA-A*0201-restricted determinant, HRP2 (74 82) (14). Consistent with these earlier observations, we observed a significant response, albeit comparatively modest in VACV-infected HHD mice and no response in HLA-A*0201/K b transgenic mice. This observation can be interpreted in a reverse manner to that posited above, namely that the presence of mouse class I leads to a decrease in the repertoire of T cells capable of recognizing the HRP2 (74 82) determinant in the context of HLA-A*0201. Identification of determinants restricted by HLA-A*1101 and HLA-B*0702 We extended this approach to characterize determinants recognized by T CD8 from HLA-A*1101/K b and HLA-B*0702/K b transgenic mice. Starting with four determinants recognized most

7 5509 FIGURE 3. CD8 T cell response against HLA-A*0201-restricted VACV epitopes. Representative experiments using different stimulator cells and purified CD8 T cells from VACVinfected HLA-A*0201/K b transgenic mice are shown. The dotted line indicates the 20 net SFCs/10 6 cells threshold used to define positivity. A and B, Representative graphs of peptides for which reactivity was seen with HLA- A*0201/K b -expressing stimulator cells, but not with target cells that lacked the HLA-A*0201 transgene. C and D, Representative graphs of peptides for which reactivity was seen with both HLA- A*0201 and H-2-expressing stimulator cells. Table I. Immunogenicity of vaccinia virus-derived HLA-A*0201-restricted peptides in different mouse strains HLA-A2.1 Potential Epitopes Peptide Sequence HLA-A2.1/K b % of peptide response Av. net SFCs/10 6 cells a HHD A 2 mice (n 1) b Av. Net SFCs/10 6 Cells a CB6F1 K b / D b / CBA/J A14L (51 59) FILGIIITV I1L ( ) RLYDYFTRV A46R ( ) GLFDFVNFV A17L (61 70) RTLLGLILFV H3L ( ) c SLSAYIIRV VACWR082 (18 26) ILDDNLYKV B14R ( ) HVDGKILFV A6L (6 14) VLYDEFVTI D12L ( ) RVYEALYYV G7L ( ) YLPEVISTI 4 66 d VACWR050 ( ) FLIVSLCPT 3 50 M1L ( ) IIIPFIAYFV A17L (81 90) ILMIFISSFL B6R ( ) LMYDIINSV HRP2 (74 82) KVDDTFYYV ND ND ND Vaccinia-infected target cells NA 1026 ND a Net SFCs/10 6 cells are calculated according to the formula: [(number of spots against relevant peptide) (number of spots against irrelevant peptides (HCV core 132)] [( )/number of effector cells/well]. b Dumped peptide at 10 g/ml. c HLA-A2-restricted peptide described by Drexler et al. (13). d ( ) indicates 20 SFCs/10 6 cells. Control-irrelevant peptides were on average 3.5 for CB6F1, 18.5 for HLA-A2.1/K b, 3.35 for K b / D b /, and 15 for CBA/J.

8 5510 POTENTIAL HLA-RESTRICTED VACCINIA VIRUS-DERIVED EPITOPES FIGURE 4. CD8 T cell response against HLA-A*1101-restricted VACV epitopes. Titration experiments using stimulator cells from different sources with purified CD8 T cells from VACV-infected HLA-A*1101/K b transgenic mice are shown. The dotted line indicates the 20 net SFCs/10 6 cells threshold used to define positivity. A, Representative graph of a peptide for which reactivity was seen with HLA- A*1101/K b -expressing stimulator cells, but not with target cells that lacked the HLA-A*1101 transgene. B, Representative graph of a peptide for which reactivity was seen with both HLA- A*1101 and H-2-expressing stimulator cells. robustly by HLA-A*1101/K b transgenic mice, we used target cells from various mice strains as well as.221-a*1101/k b -transfected target cells to determine that two determinants (I3L ( ) and E7R ( )) were restricted by HLA-A*1101 and not CB6F1 class I molecules (Fig. 4A, and data not shown). Consistent with this finding, neither of these determinants induced responses in CB6F1-VACV-infected mice. I3L ( ) induced a response in VACV-infected HLA-A*1101/K b mice that did not express mouse class I molecules (Table II). A47L ( ) and A10L ( ) exhibited a more complex restriction pattern in the experiments with different target cells. These peptides stimulated a response from HLA-A*1101/ K b -immunized mice when presented by either HLA-A*1101 or H-2 b -expressing target cells (Fig. 4B, and data not shown). Both peptides were recognized by cells from VACV-immunized CB6F1 nontransgenic mice (Table II). Overall, in terms of sensitivity, the response detected to the HLA-A*1101/K b determinants were in the 10 9 M range for peptide I3L ( ), but significantly lower (in the 10 5 to 10 7 M range) for the remaining three determinants. This relatively low avidity did not correlate with low MHC binding capacity (see below). Two of the three HLA-B*0702-restricted determinants (J2R ( ) and D1R ( )) strictly required HLA- B*0702/K b expression for antigenicity, and were not presented by CB6F1-derived LPS blasts (data not shown). The third determinant (A34R (82 90)) exhibited dual restriction in the experiments with different target cells, although the reactivity to CB6F1-pulsed target cells was relatively weak and only detected at the highest Ag dose tested (data not shown). Consistent with these findings, when the three peptides were tested in immunized HLA-B*0702/K b and wild-type CB6F1 mice, J2R ( ) and D1R ( ) were specifically recognized by HLA-B*0702/K b mice but not by CB6F1, whereas A34R (82 90) was recognized by both transgenic and nontransgenic CB6F1 mice (Table III). HLA-B*0702/K b transgenic mice that lacked expression of mouse class I molecules were not available for testing. HLA binding capacity of identified determinants It was important, of course, to test the capacity of the candidate determinants to bind to their respective class I-restricting elements (Table IV). All of the peptides bound their restricting elements with at least intermediate affinity ( 500 nm). Significantly, 12 of 14 (85.7%) -A2-, 3 of 4 (75%) -A11-, and 2 of 3 (67%) -B7-restricted determinants bound with very high affinity ( 20 nm). In the same series of experiments, the capacity of these determinants to bind to other common molecules of their supertype was determined (Table IV). In the case of the A2 supertype, 13/14 (92.9%) determinants bound a majority of the A2-supertype molecules tested, and likewise 3/4 (75%) A*1101 determinants bound Table II. Immunogenicity of vaccinia virus-derived HLA-A*1101-restricted peptides in different mouse strains Av. Net SFCs/10 6 Cells a HLA-A11 Potential Vaccinia Virus Epitopes Peptide Sequence HLA-A11/K b % of peptide response Av. net SFCs/ 10 6 cells HLA-A11/ K b / CB6F1 K b / D b / CBA/J I3L ( ) AVYGNIKHK E7R ( ) STLNFNNLR b A10L ( ) NTYLFNILYK A47L ( ) AFEFINSLLK Vaccinia-infected target cells NA a NetSFCs/10 6 cells are calculated according to the formula: [(number of spots against relevant peptide (number of spots against irrelevant peptides (HCV core 132)] [( )/number of effector cells/well]. Sample SFCs control irrelevant A11-peptide (Human Mage 3 69). NA, Not applicable. ( ) indicates 20 SFCs/10 6 cells. b Control irrelevant peptide were on average 20 SFCs/10 6 cells for HLA-A11/K b, 15 for HLA-A11/K b knockout, 15 for CB6F1; 14 for K b / D b /, and 57 for CBA/J.

9 5511 Table III. Immunogenicity of vaccinia virus-derived HLA-B*0702-restricted peptides in different mouse strains HLA-B7 Potential Epitopes Peptide Sequence HLA-B7/K b % of peptide response Av. net SFCs/ 10 6 cells a CB6F1 Av. Net SFCs/10 6 Cells a K b / D b / CBA/J A34R (82 90) LPRPDTRHL J2R ( ) KPFNNILNL b D1R ( ) RPSTRNFFEL 7 28 Vaccinia-infected target cells NA a Net SFCs/10 6 cells are calculated according to the formula: [(number of spots against relevant peptides (number of spots against irrelevant peptides (HCV core 132)] [( )/number of effector cells/well] (HBV envelope 378). b (-) indicates 20 SFCs/10 6 cells. Control irrelevant peptide were on average 6 SFCs/10 6 cells for CB6F1, 5 for HLA-B7/K b, 2 for K b / D b /, and 90 for CBA/J. a majority of the A3-supertype molecules tested. By contrast, no cross-reactivity was detected in the case of B7-supertype molecules, which selectively bound the B*0702 molecule. These results are compatible with previous reports, which highlighted a high frequency of repertoire overlap for both A2 and A3 supertypes, but a more limited overlap in the case of molecules of the B7 supertype. Table IV. HLA class I supertype binding capacity of vaccinia-derived T cell peptides Epitope A. A2-Supertype Epitopes We also analyzed these HLA determinants for the presence of H-2 binding motifs or H-2 binding capacity. In 6/6 cases of determinants exhibiting dual restriction, the peptide either contained a canonical mouse class I motif or bound with high affinity (100 nm or less) to one of the relevant purified mouse class I molecules (data not shown). HLA-A2 Supertype Binding Capacity (IC 50 nm) a A*0201 A*0202 A*0203 A*0206 A*6802 A14L (51 59) 0.11 VACWR050 ( ) 1.5 A46R ( ) 0.97 B14R ( ) M1L ( ) 9.5 VACWR082 (18 26) B6R ( ) 1.8 I1L ( ) 11 - A17L (61 70) 20 - D12L ( ) 1.5 H3L ( ) 2 - A6L (6 14) G7L ( ) 2 A17L (61 70) 22 B. A3-Supertype Peptides HLA-A3 Supertype Binding Capacity (IC 50 nm) Epitope A*1101 A*0301 A*3101 A*3301 A*6801 I3L ( ) 18 A10L ( ) 11 E7R ( ) 0.3 A47L ( ) 470 C. B7-Supertype Peptides HLA-B7 Supertype Binding Capacity (IC 50 nm) Epitope B*0702 B*3501 B*5101 B*5301 B*5401 J2R ( ) 202 D1R ( ) 11 A34R (82 90) 14 a Bold font highlights intermediate affinity binders (IC nm). A dash (-) indicates IC nm, and ( ) indicates IC nm.

10 5512 POTENTIAL HLA-RESTRICTED VACCINIA VIRUS-DERIVED EPITOPES FIGURE 5. HLA-A*0201-restricted epitopes are naturally processed by VACV-infected human cells. Epitopeimmunized CD8 T cells were stimulated with the human Jurkat HLA- A*0201/K b tumor cell line either infected with VACV at a multiplicity of infection 9, or pulsed with different concentrations of the respective peptide and their response analyzed by ex vivo ELISPOT assay. The net SFCs/10 6 CD8 T cells is shown. In conclusion, the data presented in this section supports the functional restriction data obtained from T CD8 activation assays and suggest that the A2- and A3-supertype determinants identified, Table V. VACV-WR Gene Name Structural and phylogenetic characteristics of VACWR immunogenic Ags Ag Name a Protein Size Time of Expression Description Function Category because of their high degree of cross-reactivity, could be used to elicit or monitor responses in the context of multiple HLA-class I molecules. Epitope Restriction Element % of Similarity with VAR-Major b % of Similarity with MVA b VACWR063 E7R 166 Late Soluble, myristylprotein E7R ( ) HLA-A* (E7R-VAR-IND) 100 (MVA054R) has N- myristyltransferase target VACWR094 J2R 177 Early Thymidine kinase Viral regulation J2R ( ) HLA-B* (L2R) 100 (MVA086R) VACWR106 D1R 844 Early Large subunit of mrna Viral regulation D1R ( ) HLA-B* (F1R) 90 (MVA098R) capping enzyme VACWR157 A34R 168 Late EEV glycoprotein Virion structure A34R (82 90) HLA-B* (A37R) 100 (MVA145R) VACWR172 A46R 240 Toll/IL1-receptor [TIR]-like Virulence factor A46R ( ) HLA-A* (A52R-VAR-IND; 100 (MVA159R) A49R-VAR-BSH) VACWR188 B6R 173 Ankyrin-like B6R ( ) HLA-A* (A2L) 100 (MVA174R) VACWR195 B14R 345 Early Serpin, SPI-2/CrmA Virulence factor B14R ( ) HLA-A* (B13R-VAR-IND; 55.5 (MVA025L) B12R-VAR-BSH) VACWR030 M1L 472 Early Ankyrin-like M1L ( ) HLA-A* (O1L) 50 VACWR070 I1L 312 Late Virion core DNAbinding Viral regulation I1L ( ) HLA-A* (K1L) 100 (MVA062L) protein VACWR072 I3L 269 Early ssdna-binding Viral regulation I3L ( ) HLA-A* (K3L) 100 (MVA064L) phosphoprotein VACWR085 G7L 371 Late IMV protein, proteolytic Virion structure G7L ( ) HLA-A* (H7L) 100 (MVA077L) process VACWR117 D12L 287 Early Small subunit of mrna Viral regulation D12L ( ) HLA-A* (N2L) 100 (MVA109L) capping enzyme VACWR125 A6L 372 A6L (6 14) HLA-A* (A6L-VAR-IND; 100 (MVA117L) A7L-VAR-BSH) VACWR129 A10L 891 Late Precursor p4a of core Virion structure A10L ( ) HLA-A* (A10L-VAR-IND; 100 (MVA121L) protein 4a A11L-VAR-BSH) VACWR133 A14L 90 Late IMV membrane protein Virion structure A14L (51 59) HLA-A* (A14L-VAR-IND; 100 (MVA125L) A15L-VAR-BSH) VACWR137 A17L 203 Late IMV surface membrane Virion structure A17L (61 70) HLA-A* (A17L-VAR-IND; 100 (MVA128L) protein A18L-VAR-BSH) VACWR137 A17L 203 Late IMV surface membrane Virion structure A17L (81 90) HLA-A* (A17L-VAR-IND; 100 (MVA128L) protein A18L-VAR-BSH) VACWR173 A47L 252 A47L ( ) HLA-A* (J1L) 100 (MVA160L) VACWR VACWR082 (18 26) HLA-A* (H5R) 100 (MVA074R) VACWR VACWR050 ( ) HLA-A* (C15L) 55.5 (MVA056L) VACWR101 H3L 324 Late IMV heparin binding surface protein Virion structure H3L ( ) HLA-A* (I3L) 100 (MVA093L) a Name from VACV-Cop. GenBank accession no. M b Percentage similarity of the determinant. c Percentage of similarity with ACAM3000 MVA176 (AAT10572 in GenBank protein database).

11 5513 Peptide immunization induces CD8 T cells capable of recognizing VACV-infected human cells An important potential limitation of using HLA transgenic mice is that mouse and human cells generate distinct peptide repertoires due to differences in the Ag-processing and class I assembly machineries. To address this question, we generated determinant-specific T CD8 by immunizing HLA-A*0201/K b transgenic mice with eight representative HLA-A*0201-restricted peptide. Purified T CD8 were then tested for activation against peptide-pulsed or VACV-infected APCs (Fig. 5). For all determinants tested, vigorous IFN- production was observed when VACV-infected HLA- A*0201/K b Jurkat were used as stimulator cells. This response was similar in magnitude to that elicited by HLA-A*0201/K b autologous LPS Blasts (data not shown), whereas no response was observed when CB6F1-derived stimulator cells were used (data not shown). The level of response to the VACV-infected cells was similar to that with peptide-pulsed stimulator cells, suggesting that the HLArestricted determinants identified in the HLA transgenic system are generated efficiently by natural processing in infected human cells. Nature of the Ags recognized in VACV We have identified determinants derived from 20 different VACV proteins. Table V lists for each Ag its name as defined in the Poxvirus Bioinformatics Resource Center ( (VACV protein name), as well as the name of the viral Ag according to McCraith et al. (27). Table V also lists known structural and biological characteristics of the Ags from which the determinants were derived. Among the variables listed are the predicted protein size and the stage specificity of expression (early/intermediate vs late) (27, 37 40). A description of the predicted function is also listed, when available, together with a presumed functional category (e.g., virulence factors, virion structure, viral genome regulation). Finally, the Table lists the corresponding determinants, their restriction element, and the percentage homology of the VACV sequence to that found in the genomes of variola major India and Bangladesh and modified vaccinia virus Ankara (MVA) ( The data was analyzed for significant trends, and the results are presented in Table VI. In respect to size, we found that 1 of 21 (4.7%) of the recognized ORFs were 100 residues in size, suggesting that larger Ags are more frequently recognized. In terms of stage of expression in the VACV-infectious cycle, data are available for only 15 of the recognized proteins: six are early/intermediate stage gene products, and nine are late gene products (i.e., expressed after viral DNA replication). This is remarkably concordant with known VACV gene expression (27, 37 40), where 56 gene products are believed to be expressed in early/intermediate stags and 58 gene products are expressed in late stages. In terms of the functional categories of the VACV Ags recognized, a nonrandom distribution was observed: structural proteins were most frequently recognized followed by virulence factors, with regulatory proteins being least frequently recognized ( p ). Specifically, 7 of 30 (23.3%) known structural proteins, 2 of 21 (9.52%) of reported virulence factors, and only 5 of 50 (10%) known regulatory proteins appear to be immunogenic. Our analyses included a class of putative genes, the reading frames of which overlap other larger ORFs and are annotated in the original VACV strain Copenhagen genome sequence with names such as A orf B. It is thought that the majority of these ORFs are not expressed, and our findings that of 56 such ORFs, none were found to have CTL determinants supports this contention. From the standpoint of vaccines, it is crucial to know the conservation of T CD8 determinants among the poxvirus family Table VI. Characteristics of the Ags recognized: Fisher exact statistical analysis ORFs a. Size Average Size 100 aa 100 aa Total Recognized Other Total p ORFs b. Time of Expression Time of expression Early/Intermediate Late Total Recognized Other Total p c. Function Category ORFs Virulence Factors Structural Viral Regulation Total Recognized Other Total p d. Homology of VACWR-Epitope with Variola Major India and MVA Sequences % Homology Variola MVA Total (Table VI). Of the 21 class I-restricted VACV determinants identified, 18 (85.7%) and 17 (80.9%) were identical with homologues in variola major India and MVA viruses, respectively. These results underline the potential relevance of these determinants in the context of vaccination against smallpox, or for monitoring responses resulting from immunization with MVA-based vaccines. Discussion The large coding capacity of poxvirus genomes has presented a significant challenge to the mapping of antigenic determinants. Due to technical advances on numerous fronts, it is now possible to squarely confront the complexity of diversity of the immune response to such a complex virus. We recently reported the utility of a complete cdna VACV-ORF library for discovering VACV determinants recognized by H-2 b -restricted T CD8 (28). In this study, we provide the initial demonstration of the value of a complementary approach: generation of a large panel of synthetic peptides based on in silico prediction of class I binding affinity. One pitfall of using synthetic peptide screening to identify determinants is the existence of mimotopes determinants that are antigenic but not immunogenic in the context of virus infections.

12 5514 POTENTIAL HLA-RESTRICTED VACCINIA VIRUS-DERIVED EPITOPES In most cases this arises from limitation in the Ag-processing machinery to generate the peptide from viral gene products. Mimotope cross-reactivity is a severe problem when using synthetic peptides at very high concentrations ( 10 8 M). Even highly potent peptides can represent mimotopes, however, as clearly shown by Belz et al. (41). Therefore, the epitopes identified by our study should be considered provisional until confirmed by other approaches that they are naturally processed immunogens. Despite this caveat, based on past experience with peptides demonstrating in vitro antigenicity in the nanomolar range, it is unlikely that any more than a few of peptides we identify are mimotopes. With this in mind, our findings, in conjunction with those of other studies, indicate that the T CD8 response to VACV (WR strain) does not focus on a few immunodominant proteins, but rather is spread over at least 25 different gene products, including 20 distinct VACV proteins identified in the present study in conjunction with previous reports that identified five additional immunogenic Ags (14, 28). Thus, at least 10% of possible viral gene products can be recognized by at least 1 of the 3 human class I specificities analyzed. At the same time, however, the bulk of the T CD8 response focuses on relatively few (1 4) determinants, which account for 60% of the total response in a given animal. This finding is consistent with our previous findings in C57BL/6 mice, where a single VACV determinant of five determinants identified accounts for approximately one-third of the total response to VACV (28). Our study also illustrates the complexity of immunodominance patterns. Although HLA-A*0201 serves as a restriction element for 14 determinants, only 3 4 determinants were restricted by B*0702 and A*1101. This cannot be attributed to differences in the performance of the predictive peptide binding algorithms, as determined by empirical measurements of predicted peptide binding to their respective class I molecules. Rather, this might relate, in the case of A*1101/K b transgenic mice, to the inefficient transport by mouse TAP of the ligands preferred by A*1101, which carry a C-terminalpositive charge. In the case of B*0702, the rather restrictive specificity for peptides with P in position 2 (17) resulted to only 341 predicted HLA-B7-supertype binding peptides being screened in our immunogenicity studies, as opposed to 964 HLA-A*0201- and 1584 HLA- A*1101-predicted peptides. Thus, B*0702 may simply sample a smaller pool of peptides and present proportionally less determinants. The majority of the epitopes identified in the current study are restricted by HLA-A*0201. Thus, the conclusion that responses to VACV do not focus on a few immunodominant proteins might not be correct in the case of other alleles. In fact, fewer epitopes were identified for B*0702 and A*1101 than for A*0201. One of our most striking observations is that the sum of the responses against the determinants defined is of similar magnitude to the response against virus-infected cells in each of the transgenic mouse systems investigated. This is consistent with the identification of a large portion of determinants recognized by VACVspecific T CD8 in the various mice. However, strong caveats need to be raised against this interpretation, which is probably overly simplistic. Aside from the issue of mimotopes, it is likely that the VACV-infected APCs used to measure overall responses fail to optimally present the full range of determinants presented in vivo. It is noteworthy that 5 of 14 HLA-A*0201 and 3 of 4 HLA- A*1101-restricted determinants were not recognized by T CD8 from VACV-infected HLA transgenic mice lacking the endogenous mouse MHC molecules. This suggests that the T cell repertoire is significantly influenced by nonrestricting mouse class I allomorphs. This finding has important implications for human immunity, where class I allomorphs are expressed in a large variety of permutations. In contrast to inbred mice, which express two or three class I allomorphs depending on the strain, humans express up to six distinct allomorphs of a total population of thousands of allomorphs present in appreciable frequencies in human populations. This may result in vastly different repertoires of T cells restricted by the same allomorph and, consequently, a lack of any discernable pattern of immunodominance in human responses to poxviruses. Taken together, the breadth of responses to different VACV gene products together with the variable patterns of immunodominance observed have important implications in the context of natural or nefarious immune evasion strategies. If the immune responses focused on few dominant Ags and predictable patterns of immunodominance were in place, it is conceivable that mutant or doctored viruses lacking the immunodominant Ags or determinants could result from natural evolution or be created in the laboratory. Our findings suggest that this scenario is unlikely to occur. Previously, it was reported that using inserted genes, early gene expression is associated with greater immunogenicity, possibly relating to restricted expression of early gene products in dendritic cells (42). Surprisingly, we failed to observe any bias toward early viral gene expression. Two factors could contribute to this discrepancy. First, it is possible, that the temporal control of late gene products under the control of natural viral promoters is not as stringent as the inserted gene products designed for this purpose. Second, whereas the inserted genes are excluded from virions, many of the natural late gene products are viral structural proteins, which may enter the class I-processing pathway of professional APCs during viral entry (43, 44). Indeed, we found that viral structural proteins are a preferred source of determinants. Less intuitively we found that virulence factors are also frequently recognized. This is probably related to their relatively abundant expression, because it appears that endogenous Ag-processing machinery largely samples from pools of defective ribosomal products, defective forms of viral proteins (45, 46) that are created as a byproduct of errors in protein synthesis. The poor recognition of genome regulatory proteins being consistent with this model, inasmuch as the expected low abundance of these proteins should be commensurately represented by less pools of defective ribosomal product synthesis. Our findings have important implications for diagnostic testing and vaccine development. We have identified a large number of determinants that will permit detailed experimental monitoring of cellular immune responses induced by poxvirus-specific immunization including vaccination of HLA-A2-, -A3-, and -B7-supertype-positive humans, which cover 85% (supertypes) of the human worldwide population, irrespective of ethnic background. These determinants bind their restricting element with high affinity and are candidates for the production of class I tetramer reagents, which we plan to make generally available to the scientific community. Our studies have only examined responses 7 days after immunization. In this respect it will be interesting to examine in future studies whether different epitopes are recognized at later points, or following rechallenge with VACV or heterologous viruses. The good news for vaccines is that there is high conservation of the 21 putative determinants we identify between VACV and its target pathogen VARV, and potential replacement vaccine, MVA. Future studies will address whether these determinants are recognized in humans following vaccination. Experiments are in progress with PBMC of human volunteers vaccinated with VACV or exposed to MVA. It will also be possible to use these peptides to test PBMC of individuals naturally exposed to poxviruses such as Monkeypox, responsible for a recent outbreak in the United States (6). In conclusion, our study illustrates the complex patterns of immunodominance in responses to a large virus and identifies 21 different new VACV (WR strain)-derived determinants, thus greatly expanding the number of determinants available for further study.

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Sette Derivation of HLA- B*0702 transgenic mice: functional CTL repertoire and recognition of human B*0702-restricted CTL epitopes. Hum. Immunol. 64: Alexander, J., C. Oseroff, J. Sidney, P. Wentworth, E. Keogh, G. Hermanson, F. V. Chisari, R. T. Kubo, H. M. Grey, and A. Sette Derivation of HLA- A11/Kb transgenic mice: functional CTL repertoire and recognition of human A11-restricted CTL epitopes. J. Immunol. 159: Wentworth, P. A., A. Vitiello, J. Sidney, E. Keogh, R. W. Chesnut, H. Grey, and A. Sette Differences and similarities in the A2.1-restricted cytotoxic T cell repertoire in humans and human leukocyte antigen-transgenic mice. Eur. J. Immunol. 26: Gomard, E., B. Begue, S. Sodoyer, J. L. Maryanski, B. R. Jordan, and J. P. Levy Murine cells expressing an HLA molecule are specifically lysed by HLArestricted antiviral human T cells. Nature 319: Street, M. D., T. Doan, K. A. Herd, and R. W. Tindle Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein. Immunology 106: Sesma, L., I. Alvarez, M. Marcilla, A. Paradela, and J. A. Lopez de Castro Species-specific differences in proteasomal processing and tapasin-mediated loading influence peptide presentation by HLA-B27 in murine cells. J. Biol. Chem. 278: Sidney, J., S. Southwood, D. L. Mann, M. A. Fernandez-Vina, M. J. Newman, and A. Sette Majority of peptides binding HLA-A*0201 with high affinity crossreact with other A2-supertype molecules. Hum. Immunol. 62: Sidney, J., S. Southwood, C. Oseroff, M. F. Del Guercio, A. Sette, and H. Grey Measurement of MHC/peptide interactions by gel filtration. In Current Protocols in Immunology. I. John Wiley & Sons, New York, p Sidney, J., H. M. Grey, S. Southwood, E. Celis, P. A. Wentworth, M. F. del Guercio, R. T. Kubo, R. W. Chesnut, and A. 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Sette Structural features of peptide analogs of human histocompatibility leukocyte antigen class I epitopes that are more potent and immunogenic than wild-type peptide. J. Exp. Med. 194: Sette, A., A. Vitiello, B. Reherman, P. Fowler, R. Nayersina, W. M. Kast, C. J. Melief, C. Oseroff, L. Yuan, J. Ruppert, et al The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J. Immunol. 153: Sette, A., and J. Sidney Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50: Boon, T., and A. Van Pel T cell-recognized antigenic peptides derived from the cellular genome are not protein degradation products but can be generated directly by transcription and translation of short subgenic regions: a hypothesis. Immunogenetics 29: Price, N., D. C. Tscharke, M. Hollinshead, and G. L. Smith Vaccinia virus gene B7R encodes an 18-kDa protein that is resident in the endoplasmic reticulum and affects virus virulence. Virology 267: Price, N., D. C. Tscharke, and G. L. Smith The vaccinia virus B9R protein is a 6 kda intracellular protein that is non-essential for virus replication and virulence. J. Gen. Virol. 83: Betakova, T., and B. Moss Disulfide bonds and membrane topology of the vaccinia virus A17L envelope protein. J. Virol. 74: Senkevich, T. G., B. M. Ward, and B. Moss Vaccinia virus A28L gene encodes an essential protein component of the virion membrane with intramolecular disulfide bonds formed by the viral cytoplasmic redox pathway. J. Virol. 78: Belz, G. T., P. G. Stevenson, and P. C. Doherty Contemporary analysis of MHC-related immunodominance hierarchies in the CD8 T cell response to influenza A viruses. J. Immunol. 165: Restifo, N. P., I. Bacik, K. R. Irvine, J. W. Yewdell, B. J. McCabe, R. W. Anderson, L. C. Eisenlohr, S. A. Rosenberg, and J. R. Bennink Antigen processing in vivo and the elicitation of primary CTL responses. J. Immunol. 154: Yewdell, J. W., J. R. Bennink, and Y. Hosaka Cells process exogenous proteins for recognition by cytotoxic T lymphocytes. Science 239: Riddell, S. R., M. Rabin, A. P. Geballe, W. J. Britt, and P. D. Greenberg Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression. J. Immunol. 146: Yewdell, J. W., L. C. Anton, and J. R. Bennink Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules? J. Immunol. 157: Princiotta, M. F., D. Finzi, S. B. Qian, J. Gibbs, S. Schuchmann, F. Buttgereit, J. R. Bennink, and J. W. Yewdell Quantitating protein synthesis, degradation, and endogenous antigen processing. Immunity 18:

14 CORRECTIONS Joshi, P. C., L. Applewhite, J. D. Ritzenthaler, J. Roman, A. L. Fernandez, D. C. Eaton, L. A. S. Brown, and D. M. Guidot Chronic ethanol ingestion in rats decreases granulocyte-macrophage colony-stimulating factor receptor expression and downstream signaling in the alveolar macrophage. J. Immunol. 175: In Figure 1, panel C was omitted. The corrected figure is shown below. The error has been corrected in the online version, which now differs from the print version as originally published. Copyright 2005 by The American Association of Immunologists, Inc /05/$02.00

15 8440 CORRECTIONS Li, X., K. Malathi, O. Krizanova, K. Ondrias, K. Sperber, V. Ablamunits, and T. Jayaraman Cdc2/cyclin B1 interacts with and modulates inositol 1,4,5-trisphosphate receptor (type 1) functions. J. Immunol. 175: In the author line, the sequence of the first two authors is reversed. The corrected author line is shown below. Krishnamurthy Malathi, Xiaogui Li, Olga Krizanova, Karol Ondrias, Kirk Sperber, Vitaly Ablamunits, and Thottala Jayaraman Pasquetto, V., H.-H. Bui, R. Giannino, F. Mirza, J. Sidney, C. Oseroff, D. C. Tscharke, K. Irvine, J. R. Bennink, B. Peters, S. Southwood, V. Cerundolo, H. Grey, J. W. Yewdell, and A. Sette HLA-A*0201, HLA-A*1101, and HLA- B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products. J. Immunol. 175: The fourth author s name, Cindy Banh, was omitted. The correct list of authors and affiliations is shown below. Valerie Pasquetto,* Huynh-Hoa Bui,* Rielle Giannino,* Cindy Banh,* Fareed Mirza, John Sidney,* Carla Oseroff,* David C. Tscharke, Kari Irvine, Jack R. Bennink, Bjoern Peters,* Scott Southwood, Vincenzo Cerundolo, Howard Grey,* Jonathan W. Yewdell, and Alessandro Sette 2* *La Jolla Institute for Allergy and Immunology, San Diego, CA 92109; Tumor Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford, United Kingdom; Epimmune Incorporated, San Diego, CA 92121; Laboratory of Viral Diseases, National Institutes of Health, Bethesda, MD 20892; and Division of Immunology and Infectious Diseases, Queensland Institute of Medical Research, Herston, Queensland, Australia Zhang, X., P. Shan, S. Qureshi, R. Homer, R. Medzhitov, P. W. Noble, and P. J. Lee Cutting edge: TLR4 deficiency confers susceptibility to lethal oxidant lung injury. J. Immunol. 175: In Materials and Methods, in the first sentence under the heading Intranasal administration of recombinant adenovirus-containing HO-1 cdna, the source for adenoviral HO-1 cdna was incorrectly attributed. The source is stated in the corrected sentence below. Mice were anesthetized with methoxyflurane, and then PFU of adenoviral HO-1 (Ad-HO-1) (a gift from K. Kolls, University of Pittsburgh Medical Center, Pittsburgh, PA, and J. Alam, Alton Ochsner Medical Foundation, New Orleans, LA) (29) or adenoviral -galactosidase (Ad-LacZ) (BD Biosciences) were administered intranasally to each mouse in a volume of 50 l as described previously (12). The authors also wish to add the reference shown below. 29. Otterbein, L. E., J. K. Kolls, L. L. Mantell, J. L. Cook, J. Alam, and A. M. K. Choi Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J. Clin. Invest. 103:

16 8441 Gays, F., K. Martin, R. Kenefeck, J. G. Aust, and C. G. Brooks Multiple cytokines regulate the NK gene complexencoded receptor repertoire of mature NK cells and T cells. J. Immunol. 175: In Figure 1, a sentence regarding the solid and broken lines was omitted from the legend. The corrected legend is shown below. FIGURE 1. Specificity of the CM4 mab. A, YB2 or RNK cells transfected with Ly49 constructs were stained with medium or first layer Abs followed by AF488 goat anti-mouse Ig. Solid lines: staining by CM4. Left broken line: medium control. Right broken line: staining by positive control Abs Ly49A A1, Ly49B 1A1, Ly49C 4D12, Ly49D 4E5, Ly49E 4D12, Ly49F HBF, Ly49G 4G11, Ly49H 3D10, Ly49I YBI. B, Cross-competition between Abs. YB2 cells transfected with Ly49E (YB2-E) and RNK cells transfected with Ly49F (RNK-F) were incubated with medium or saturating quantities of the unlabeled Ly49 Abs shown on the y-axis. After 20 min, AF488-labeled CM4, 4D12, or HBF Ab was added, and incubation was continued for an additional 20 min. Median fluorescence values were determined by flow cytometry, and the percentage inhbition caused by pretreatment with each unlabeled Ab is plotted on the y-axis. The likelihood that the inhibition observed was due to chance variation was determined by Student s t test (*, p 0.05, **, p 0.01, ***, p 0.001). The experiments shown are representative of three similar experiments of each type that were performed. In Figure 9A, the gel image labeled Ly49A is inverted. The corrected figure is shown below.

17 8442 CORRECTIONS Rakoff-Nahoum, S., H. Chen, T. Kraus, I. George, E. Oei, M. Tyorkin, E. Salik, P. Beuria, and K. Sperber Regulation of class II expression in monocytic cells after HIV-1 infection. J. Immunol. 167: Figure 10, demonstrating intracellular trafficking of HLA-DR after the introduciton of HIV proteins, is incorrect. The corrected figure is shown below. Lukacs, N. W., K. K. Tekkanat, A. Berlin, C. M. Hogaboam, A. Miller, H. Evanoff, P. Lincoln, and H. Maassab Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms. J. Immunol. 167: In Materials and Methods, in the first sentence under the heading RSV infection, the designation of the virus type should be human RSV A strain, not A2 strain. Tekkanat, K. K., H. F. Maassab, D. S. Cho, J. J. Lai, A. John, A. Berlin, M. H. Kaplan, and N. W. Lukacs IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent. J. Immunol. 166: In Materials and Methods, in the first sentence under the heading Virus and infection, the designation of the virus type should be human RSV A strain, not A2 strain.