Effective circovirus vaccines require minimal numbers of conserved B and T cell epitopes that provide protective immunity. At the same time, vaccines must have the capacity to distinguish infected from vaccinated animals (DIVA). Accomplishing both goals requires knowledge of critical B and T cell epitopes. Current efforts in vaccine development have primarily focused on B cell epitopes with little information on the location and composition of T cell epitopes that offer helper function. This proposal had the objective of identifying protective T cell epitopes in the capsid protein of PCV2. The identification of protective T cell epitopes was predicated on the fact that the commercial vaccines have been shown to protect pigs from disease and to reduce viral load. Pigs that were PCV2 negative by PCR assay were vaccinated at 3 and 6 weeks of age with a commercially available baculovirus expressed ORF2 vaccine according to label instructions. Three weeks later pigs were necropsied and lymph node cells collected and assayed. This collection time approximates the exposure of pigs to PCV2 in the finisher. Synthesized PCV2a subtype capsid peptides of 30 amino acid residues each were used in two types of assays: the ELISPOT assay to enumerate interferon-gamma secreting cells, and a very sensitive flow cytometric proliferation assay using a fluorescent dye to label cells prior to stimulation in order to determine what proportion responded to a given stimulant. Proliferation by CD4+ (helper) T cells was determined in the proliferation assay by labeling cells after culture with antibody to the CD4 molecule followed by a fluorescent secondary antibody, and directly determining the proportion of CD4+ T cells that proliferated. The results from this project demonstrated a specific pattern of interferon-gamma secretion in response to individual peptides occurred from T lymphocytes from vaccinated pigs, but not from vaccine control pigs. The pattern was bimodal with the greatest responses to the N-terminal and C-terminal portions of the capsid protein. A similar pattern occurred in the CD4+ T cell proliferation assay, although the distribution was less bimodal with responses to more of the peptides compared with the secretion assay. Vaccine controls tended not to have CD4+ T cells that responded to the peptides in the proliferation assay. These data support the idea that the PCV2 capsid protein contains protective T cell epitopes that stimulate CD4+ T cells. The information is important because it provides the basis for establishing a protective vaccine that includes protective T cell epitopes. Contact: Carol Wyatt, cwyatt@vet.k-state.edu