Abstract
The aims were to identify potential immunodominant CD4 T-cell antigens, estimate within-subject CD4 diversity, and investigate CD4 T-B cell concurrence. Each of 205 predicted vaccinia ORFs was transcribed and translated in vitro. Serial PBMC and sera were obtained from vaccinated subjects. Antibody binding assays used protein-spotted arrays. CD4 proliferation assays used bulk PBMC-derived responders and autologous APC. Overall, CD4 and antibody responses were above background for 145 (70%) and 62 (30%) ORFs, respectively. The mean number of ORFs recognized per specimen by CD4 cells was 55 (range, 20 to 83). The ORFs recognized most frequently by CD4 cells were L4R, D13L, A10L, I3L, H3L, WR149, WR148, and A3L, while for antibodies, the most frequently detected responses were to A10L, B2R, H3L, WR148, I1L, D13L, D8R, and H5R. The CD4 immunodominant ORFs were mostly virion structural proteins and had late expression kinetics during virus replication. Each ORF immunodominant for antibody responses also frequently elicited CD4 responses, while antibody responses were not detected to many other CD4 antigens. This is the first proteome-wide screen of CD4 responses to a complex pathogen using full-length protein antigens. Immunodominant CD4 antigens identified using this technology may be rational subunit vaccine components for microbial pathogens requiring T-cell help for protection. First two authors contributed equally to the work.
