Identification of Murine Poxvirus-Specific CD8  CTL Epitopes with Distinct Functional Profiles 1 Anuja Mathew, 2 Masanori Terajima, Kim West, Sharone Green, Alan L. Rothman, Francis A. Ennis, and Jeffrey S. Kennedy Murine T cell epitopes against vaccinia virus (VV) have not been characterized to date in part due to the large and complex genome of VV. We have identified and characterized two CD8  T cell epitopes on the A47L (modified VV Ankara strain (MVA)-029) and J6R (MVA-043) proteins of VV that are D b and K b restricted, respectively. Following i.p. immunization with VV New York City Board of Health (NYCBH) strain, MVA-029 peptide-stimulated splenocytes secreted IFN- from 7 days to 7 mo postimmunization, and virus-stimulated effectors were also able to lyse MVA-029-pulsed target cells at the same time points. In contrast, MVA-043 peptide-stimulated splenocytes secreted very low levels of IFN- only at day 7 but maintained the ability to lyse target cells up to 2 mo postimmunization. Both MVA-029 and MVA-043 peptide-stimulated lymph node cells degranulated similarly as assessed by Ag-induced CD107 expression. T cell responses to whole-virus stimulation remained robust and steady during the acute and memory T cell response to VV. Identification of T cell epitopes on VV will enable further studies to increase our understanding of the role of CD8  T cells in VV infection and assist in the design of new protective strategies. The Journal of Immunology, 2005, 174: 2212–2219. V accinia virus (VV) 3 is a large DNA virus and is a mem- ber of the Poxviridae family. It has been used as an effective vaccine against variola virus, the causative agent of smallpox. VV has also been used extensively as an ex- pression vector for foreign genes, as a recombinant vaccine, and has been the most widely studied member in its family (1). Despite the use of VV in numerous experimental systems, study of the immune response to the parent vector has been limited. Recently, the immunologic correlates of protection following infection with different strains of VV have begun to be examined. Both cellular and humoral immunity are thought to play a role in protection against orthopoxviruses (2, 3). NK cells and  T cells play a role in innate resistance to infection (4, 5). In a study per- formed by Xu et al. (2), CD4 T cells and neutralizing Abs (nAb) were important for clearing acute VV infection, whereas CD8 T cells seemed to play a less dominant role during the acute phase of infection. However, Karupiah et al. (6) have shown that CD8  T cells are essential for recovery from infection with the poxvirus ectromelia virus. Additionally, in humans, the presence of nAb does not prevent the development of progressive vaccinia if cell- mediated immunity is defective (7). Xu et al. have also shown that, in the absence of Ab and CD4 T cells, CD8 T cells were necessary for protection, because skin lesions and adrenal necrosis were ex- acerbated in MHC class II / CD8-depleted mice. Adoptive trans- fer experiments have shown that CD8  T cells can mediate control of VV infection (2). Recently, we defined two HLA-A*0201-restricted poxvirus cross-reactive CD8  T cell epitopes designated 74A (C16L aa 79 – 87) and 165 (C7L aa 74 – 82) (8). HLA-A*0201 transgenic mice immunized with peptide 165 were shown by Snyder et al. (9) to protect mice from a lethal dose of VV WR strain intranasally, indicating that CD8  CTLs directed against a single epitope are protective. These data indicate that multiple arms of the effector immune response are likely involved in the acute and memory phase of VV infection and point to an important role for CD8 T cells in mediating protection and recovery from infection. Following immunization of mice with VV, a robust T cell re- sponse is elicited with activated VV-specific CD8 T cells and CD4 T cells (10). By 2–3 wk, a very stable but lower virus-specific response is maintained long term in both C57BL/6 as well as BALB/c mice (2, 10). Although numerous proteins that elicit nAb responses have been mapped, no murine T cell epitopes have been defined against VV. The absence of defined murine CD8 T cell epitopes against VV has limited functional studies to date. We identified a D b - and a K b -restricted CD8 T cell epitope on the A47L and J6R proteins of VV. Following immunization with VV New York City Board of Health (NYCBH), virus-stimulated effectors were able to lyse virus-infected as well as peptide-pulsed targets efficiently during both the acute and memory phases of infection. Although peptide MVA-029 stimulated large numbers of IFN--secreting cells through all phases of the immune response, peptide MVA-043-stimulated splenocytes showed significantly lower numbers of IFN--producing cells at 7 days postimmuniza- tion, and by day 14 IFN--secreting cells were undetectable. How- ever, Ag-induced CD107a expression did not differ between the two epitopes. Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, MA 01655 Received for publication August 24, 2004. Accepted for publication November 23, 2004. 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 project has been funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under Con- tract No. N01-AI-25490 and Grant U19 AI057319. 2 Address correspondence and reprint requests to Dr. Anuja Mathew, Center for In- fectious Disease and Vaccine Research, S5-326, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: anuja.mathew@umassmed.edu 3 Abbreviations used in this paper: VV, vaccinia virus; nAb, neutralizing Ab; NYCBH, VV New York City Board of Health; BMDC, bone marrow-derived den- dritic cell; MVA, modified VV Ankara strain; MOI, multiplicity of infection; ICS, intracellular cytokine staining; LN, lymph node. The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00