Dominance and Diversity in the Primary Human CD4 T Cell Response to Replication-Competent Vaccinia Virus 1 Lichen Jing,* Tiana M. Chong, † Benjamin Byrd,* Christopher L. McClurkan, † Jay Huang, † Brian T. Story, † Karissa M. Dunkley, † Lydia Aldaz-Carroll, ‡ Roselyn J. Eisenberg, ‡§ Gary H. Cohen, ‡ William W. Kwok, ¶ Allesandro Sette,  David M. Koelle 2 * †¶# ** Vaccination with replication-competent vaccinia protects against heterologous orthopoxvirus challenge. CD4 T cells have essential roles helping functionally important Ab and CD8 antiviral responses, and contribute to the durability of vaccinia-specific memory. Little is known about the specificity, diversity, or dominance hierarchy of orthopoxvirus-specific CD4 T cell responses. We interrogated vaccinia-reactive CD4 in vitro T cell lines with vaccinia protein fragments expressed from an unbiased genomic library, and also with a panel of membrane proteins. CD4 T cells from three primary vaccinees reacted with 44 separate antigenic regions in 35 vaccinia proteins, recognizing 8 to 20 proteins per person. The integrated responses to the Ags that we defined accounted for 49 to 81% of the CD4 reactivity to whole vaccinia Ag. Individual dominant Ags drove up to 30% of the total response. The gene F11L-encoded protein was immunodominant in two of three subjects and is fragmented in a replication- incompetent vaccine candidate. The presence of protein in virions was strongly associated with CD4 antigenicity. These findings are consistent with models in which exogenous Ag drives CD4 immunodominance, and provides tools to investigate the relation- ship between Ab and CD4 T cell specificity for complex pathogens. The Journal of Immunology, 2007, 178: 6374 – 6386. V accinia viruses confer long-term protection from variola. Poxviruses are being used to deliver heterologous Ags in the settings of vaccines and immunotherapy. CD4 T cells likely are important in primary clearance of vaccinia and in the induction and maintenance of long-term memory and protec- tion from variola challenge. We conducted proteome-wide screens of the CD4 response shortly after primary immunization. CD4 T cells help generate functionally important CD8 re- sponses (1) through licensing of DC via CD40L (2). Helper functions include the priming of naive CD8 responses, the tran- sition of primary CD8 cells into memory, the survival of CD8 memory cells, and the generation of secondary CD8 responses in specific models (3). CD4 help for CD8s is likely needed at one or more points for most viruses (4). This is especially true if viral immune evasions dampen innate signals that can mature DC, as does vaccinia (5–7). In vivo, CD4-depleted, CD4 -/- , and MHC II -/- mice show delayed viral clearance and increased mortality after vaccinia in- fection (8, 9). Modified vaccinia Ankara (MVA) 3 fails to protect MHC II -/- mice against vaccinia challenge (9). Vaccinia-specific CD8 T cells in MHC II -/- or CD4 -/- mice have proliferative and functional protection defects (10 –12) and cannot transition into memory cells (4). Ectromelia virus is an orthopoxvirus that infects mice in nature. Although primary CD8 responses to ectromelia virus are normal in CD4-depleted mice, helpless CD8 cells do not transition into memory or reactivate upon challenge. CD4-depleted mice fail to clear EV (13, 14). CD4 T cells assist Ag-specific Ab synthesis. Orthopoxvirus- specific Abs are necessary and sufficient for vaccine-induced pro- tection from orthopoxvirus challenge (15, 16). CD4-deficient mice do not make specific Abs (8, 9). The cognate T-B help theory (17) predicts that targets of specific Abs should elicit CD4 responses. IFN-, TNF-, and cytotoxicity are additional antiviral functions of human vaccinia-specific CD4 T cells (18 –21). Human data are consistent with important roles for CD4 T cells. The estimated half-life for CD4 responses to vaccinia is 8 –15 years (18, 22, 23). Among donors in the decay phase of memory responses, persons with intact CD8 responses always had detect- able CD4 responses (18, 24). Persons with HIV-related or idio- pathic CD4 deficiency may fail to clear poxvirus infection (25). The specificity of CD4 responses to vaccinia is largely unknown. To probe the entire proteome in an unbiased fashion, we used a genomic DNA library based on a replication-competent vaccinia strain. We report that the CD4 response is very diverse, targeting up to 20 proteins per subject, and can be placed in a unique hierarchy for each person including immunodominant responses. The response em- phasizes virion structural proteins and enzymes involved in viral rep- lication. Detection of CD4 responses is highly correlated with the presence of the protein in purified virion preparations. These data are *Department of Medicine, † Department of Laboratory Medicine, University of Washington, Seattle, WA 98101; ‡ School of Veterinary Medicine, § School of Dental Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; ¶ Benaroya Research Institute, Seattle, WA 98101;  La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; # Fred Hutchinson Cancer Research Center, Seattle, WA 98109; **Program in Pathobiology, University of Washington, Seattle, WA 98101 Received for publication January 3, 2007. Accepted for publication February 27, 2007. 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 National Institutes of Health Grants AI061636 and AI067496 (to D.M.K.), AI062486 and AI057168 (to G.H.C. and R.J.E.), and AI56268 and HHSN266200400124C (to A.S.). 2 Address correspondence and reprint requests to Dr. David Koelle, 1616 Eastlake Avenue E., Suite 500, Box 358117, Seattle, WA 98102. E-mail addresss: viralimm@ u.washington.edu 3 Abbreviations used in this paper: MVA, modified vaccinia Ankara; ICC, intracel- lular cytokine cytometry; WR, Western Reserve; TCM, T cell medium; ORF, open reading frame. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org